EA011876B1 - ANTIBODIES TO IFN-γ AND METHOD FOR PRODUCING AND USE THEREOF - Google Patents

Abstract

This invention provides antibodies that interact with or bind to human interferon-gamma (IFN-gamma) and methods for treating IFN-gamma mediated diseases by administering a pharmaceutically effective amount of antibodies to IFN-gamma. Methods of detecting the amount of IFN-gamma in a sample using antibodies to IFN-gamma are also provided.

Description

The invention relates to human monoclonal antibodies that bind interferon-gamma (ΙΡΝ-γ). Compositions and methods for treating заболеваний-γ mediated diseases are also described.

BACKGROUND OF THE INVENTION

Interferons (ΙΡΝ) were originally named so for their ability to prevent (from the English. I. and G. Gege) viral infection of the host cells (Jaask aib Lbebeptap, 1957, Prg. B. 8c. 147: 28-267). After their discovery, a number of members of the interferon family were identified having various biological functions in addition to antiviral protection, including cell growth and cellular immunity. Interferons of types ΙΡΝ-α, ΙΡΝ-β, ΙΡΝ-ω and ΙΡΝ-τ are type I interferons and bind the ΙΡΝ type тор receptor, while ΙΡΝ-γ is the фер type interferon and binds the ΙΡΝ type ΙΙ receptor (Reeyeg e! A1 ., 1998, Sapseg Century. 58: 2489-2499).

ΙΡΝ-γ signal transmission depends on at least five different proteins: ΙΡΝΟΒ1 and ΙΡΝΟΒ2 (subunits of the ΙΡΝ-γ receptor), 1a1, 1a2, and the transcription factor 8TLT1 (8cBnb1b apb Eagpe11. 1995, Appi. Veu. Vusyet. 64: 621-651 ; Vasya e! A1., 1997, Appi. Veu. Ittipo.1. 15: 563-591). ΙΡΝ-γ receptors have been found on most types of cells, with the exception of mature red blood cells (Raggag apb sigréb, 1993, Appi. Veu. Itipo1. 11: 571-611). Proteins 1AK1, 1AK2 and 8TAT1 mediate передачу-γ signal transmission.

ΙΡΝ-γ regulates various biological functions, such as antiviral responses, cell growth, immune response and tumor suppression, and ΙΡΝ-γ can be involved in various human diseases. Thus, in this area there is a need for agents that can modulate the biological activity of ΙΡΝ-γ.

Summary of the invention

The invention provides monoclonal antibodies that bind to interferon-γ (ΙΡΝ-γ), and polynucleotides encoding them. These antibodies can inhibit or modulate at least one of the biological activities of ΙΡΝ-γ and can be useful to mitigate the effects of ΙΡΝ-γ-mediated diseases. The invention also provides hybridoma cells that produce and can secrete the monoclonal antibodies of the invention into cell culture media. Antibodies of the invention may be useful in the treatment of,-γ mediated diseases.

In some aspects of the invention, antibodies are provided, possibly monoclonal antibodies and / or human antibodies, which may comprise a heavy chain and a light chain, where the heavy chain includes the amino acid sequence defined in 8EO ΙΌ ΝΟ: 2, or an antigen-binding or immunologically functional immunoglobulin fragment thereof.

In some aspects of the invention, antibodies are provided, possibly monoclonal antibodies, which may be human antibodies comprising a heavy chain and a light chain, where the heavy chain includes a constant region of the heavy chain of Ι§Ο1, Ι§Ο2 or Ι§Ο4. In some embodiments, an antibody of the invention comprises the amino acid sequence of the constant region of the heavy chain Ι§Ι1, as defined in 8EO ΙΌ ΝΟ: 2, or an antigen-binding or immunologically functional immunoglobulin fragment thereof.

In some aspects, the antibodies of the invention comprise a heavy chain and a light chain, where the variable region of the heavy chain includes an amino acid sequence defined in any of 8EO ΙΌ ΝΟ: 6, 8EO ΙΌ ΝΟ: 10, 8EO ΙΌ ΝΟ: 14 or 8EO ΙΌ ΝΟ: 30, or its antigen-binding or immunologically functional immunoglobulin fragment. In other aspects, the light chain variable region includes an amino acid sequence defined in any of 8EO ΙΌ ΝΟ: 8, 8EO ΙΌ ΝΟ: 12, 8EO ΙΌ ΝΟ: 16 or 8EO ΙΌ ΝΟ: 31, or an antigen-binding or immunologically functional immunoglobulin fragment thereof. In additional aspects, the heavy chain includes an amino acid sequence defined in any of 8EO ΙΌ ΝΟ: 17, 8EO ΙΌ ΝΟ: 19, 8EO ΙΌ ΝΟ: 21 or 8EO ΙΌ ΝΟ: 32, or an antigen-binding or immunologically functional immunoglobulin fragment thereof. According to other aspects, the light chain includes an amino acid sequence defined in any of 8EO ΙΌ ΝΟ: 18, 8EO ΙΌ ΝΟ: 20, 8EO ΙΌ ΝΟ: 22 or 8EO ΙΌ ΝΟ: 33, or an antigen-binding or immunologically functional immunoglobulin fragment thereof.

The invention also provides antibodies that specifically bind to ΙΡΝ-γ, in which the heavy chain comprises the variable region of the heavy chain containing the amino acid sequence defined in 8EO ΙΌ ΝΟ: 6, or an antigen-binding or immunologically functional immunoglobulin fragment thereof, and the light chain includes a variable a light chain region containing the amino acid sequence defined in 8EO ΙΌ ΝΟ: 8, or an antigen-binding or immunologically functional immunoglobulin fragment thereof.

Antibodies and immunologically functional immunoglobulin fragments thereof which can specifically bind to γ-γ and / or inhibit or modulate the biological activity of γ-γ containing a heavy chain and light are also provided in certain aspects of the invention.

- 1 011876 chain, where the heavy chain includes the variable region of the heavy chain, and the variable region of the heavy chain includes a sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or about 99% identity with the amino acid sequence defined in 8EO ΙΌ N0: 6, and where the light chain includes the variable region of the light chain, and the variable region of the light chain includes a sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identity with the amino acid sequence, defined in 8E0 ΙΌ N0: 8, where this antibody interacts with ΙΕΝ-γ.

In addition, the invention provides antibodies that can inhibit or modulate biological activity and / or specifically bind to ΙΕΝ-γ, where the heavy chain includes the variable region of the heavy chain containing the amino acid sequence defined in 8E0 ΙΌ N0: 10, or its antigen-binding or immunologically functional immunoglobulin fragment, and the light chain includes the variable region of the light chain containing the amino acid sequence defined in 8E0 ΙΌ N0: 12, or its antigen-binding il immunologically functional immunoglobulin fragment thereof.

In some aspects of the invention, antibodies are provided that can inhibit or modulate biological activity and / or specifically bind to IΕN-γ containing a heavy chain and a light chain, where the heavy chain comprises a variable region of a heavy chain, this variable region of a heavy chain comprising a sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identity with the amino acid sequence defined in 8E0 ΙΌ N0: 10, and where the light chain includes the variable region of the light tse pi, this variable region of the light chain comprising a sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identity with the amino acid sequence defined in 8E0 ΙΌ N0: 12, moreover, this antibody interacts with IΕN-γ.

In addition, the invention provides antibodies that can inhibit or modulate biological activity and / or specifically bind to IΕN-γ, where the heavy chain includes the variable region of the heavy chain containing the amino acid sequence defined in 8E0 ΙΌ N0: 30, or its antigen-binding or immunologically functional immunoglobulin fragment, and the light chain includes the variable region of the light chain containing the amino acid sequence defined in 8E0 ΙΌ N0: 12, or its antigen-binding or immunologically functional immunoglobulin fragment.

In some aspects of the invention, antibodies are provided that can inhibit or modulate biological activity and / or specifically bind to IΕN-γ containing a heavy chain and a light chain, where the heavy chain includes the variable region of the heavy chain, and where the variable region of the heavy chain includes a sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identity with the amino acid sequence defined in 8E0 ΙΌ N0: 30, and where the light chain includes the variable region of the light chains, and the light chain variable region comprises a sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identity with the amino acid sequence defined in 8E0 ΙΌ N0: 12 where this antibody interacts with IΕN-γ.

The invention also provides antibodies that can inhibit or modulate biological activity and / or specifically bind to IΕN-γ, where the heavy chain includes the variable region of the heavy chain containing the amino acid sequence defined in 8E0 ΙΌ N0: 14, or its antigen-binding or immunologically functional immunoglobulin fragment, and the light chain includes the variable region of the light chain containing the amino acid sequence defined in 8E0 ΙΌ N0: 16, or its antigen-binding or immun a physiologically functional immunoglobulin fragment.

In some aspects of the invention, antibodies are provided that can specifically bind to IΕN-γ and / or inhibit or modulate the biological activity of IΕN-γ containing a heavy chain and a light chain, where the heavy chain includes the variable region of the heavy chain, and this variable region of the heavy chain includes a sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identity with the amino acid sequence defined in 8E0 ΙΌ N0: 14, and where the light chain includes variable region easily chain, and this variable region of the light chain includes an amino acid sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identity with the amino acid sequence defined in 8E0 ΙΌ N0: 16, moreover, this antibody interacts with IΕN-γ.

The invention also provides antibodies that can inhibit or modulate biological activity and / or specifically bind to IΕN-γ, where the heavy chain includes the variable region of the heavy chain containing the amino acid sequence defined in 8E0 ΙΌ N0: 14, or its antigen-binding or immunologically functional immunoglobulin fragment, and the light chain includes the variable region of the light chain containing the amino acid sequence defined in 8E0 ΙΌ N0: 31, or its antigen-binding or immun biological function

- 2 011876 local immunoglobulin fragment.

In some aspects of the invention, antibodies are provided that can inhibit or modulate biological activity and / or specifically bind to β-γ containing a heavy chain and a light chain, where the heavy chain comprises a variable region of a heavy chain, this variable region of a heavy chain comprising a sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identity with the amino acid sequence defined in ZEC ΙΌ N0: 14, and where the light chain includes the variable region of the lightchain, and this variable region of the light chain includes an amino acid sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identity with the amino acid sequence defined in ZEC ΙΌ N0: 31, moreover, this antibody interacts with ΙΡΝ-γ.

The invention also provides antibodies that can inhibit or modulate biological activity and / or specifically bind to ΙΡΝ-γ, where the heavy chain includes the amino acid sequence defined in ZEC ΙΌ N0: 17, or its antigen-binding or immunologically functional immunoglobulin fragment, and the light chain includes the amino acid sequence defined in 8EC ΙΌ N0: 18, or its antigen-binding or immunologically functional immunoglobulin fragment.

In some aspects of the invention, antibodies are provided that can inhibit or modulate biological activity and / or specifically bind to IΡN-γ containing a heavy chain and a light chain, where the heavy chain comprises a variable region of a heavy chain, this variable region of a heavy chain comprising a sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identity with the amino acid sequence defined in ZEC ΙΌ N0: 17, and where the light chain includes the variable region of the light c epi, wherein this variable region of the light chain comprises an amino acid sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identity with the amino acid sequence defined in the EC ΙΌ N0: 18, moreover, this antibody interacts with IΡN-γ.

In addition, the invention provides antibodies that can inhibit or modulate biological activity and / or specifically bind to IΡN-γ, where the heavy chain includes the amino acid sequence defined in ZEC N0: 19, or its antigen-binding or immunologically functional immunoglobulin fragment, and the light chain includes the amino acid sequence defined in ZEC ΙΌ N0: 20, or its antigen-binding or immunologically functional immunoglobulin fragment.

In some aspects of the invention, antibodies are provided that can inhibit or modulate biological activity and / or specifically bind to IΡN-γ containing a heavy chain and a light chain, where the heavy chain comprises a variable region of a heavy chain, this variable region of a heavy chain comprising a sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identity with the amino acid sequence defined in ZEC ΙΌ N0: 19, and where the light chain includes the variable region of the light c epi, wherein this variable region of the light chain comprises an amino acid sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identity with the amino acid sequence defined in the EC ΙΌ N0: 20, where this antibody interacts with IΡN-γ.

The invention also provides antibodies that can inhibit or modulate biological activity and / or specifically bind to IΡN-γ, where the heavy chain includes the amino acid sequence defined in ZEC ΙΌ N0: 21, or its antigen-binding or immunologically functional immunoglobulin fragment, and the light chain includes the amino acid sequence defined in ZEC ΙΌ N0: 22, or its antigen-binding or immunologically functional immunoglobulin fragment.

In some aspects of the invention, antibodies are provided that can inhibit or modulate biological activity and / or specifically bind to ΛΉ-γ containing a heavy chain and a light chain, where the heavy chain comprises a variable region of a heavy chain, and this variable region of a heavy chain includes a sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identity with the amino acid sequence defined in ZEC ΙΌ N0: 21, and where the light chain includes the variable region of the light c epi, wherein this variable region of the light chain comprises an amino acid sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identity with the amino acid sequence defined in the EC ΙΌ N0: 22, moreover, this antibody interacts with IΡN-γ.

The invention also provides antibodies that can inhibit or modulate biological activity and / or specifically bind to IΡN-γ, where the heavy chain includes the amino acid sequence defined in ZEC ΙΌ N0: 32, or its antigen-binding or immunologically functional immunoglobulin fragment, and the light chain includes the amino acid sequence defined in ZEC ΙΌ N0: 20, or its antigen-binding or immunologically functional immunoglobulin fragment.

- 3 011876

In some aspects of the invention, antibodies are provided that can inhibit or modulate biological activity and / or specifically bind to β-γ containing a heavy chain and a light chain, where the heavy chain includes the variable region of the heavy chain, and where the variable region of the heavy chain includes a sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identity with the amino acid sequence defined in 8EO ΙΌ N0: 32, and where the light chain includes the light variable region chains where the variable region of the light chain includes an amino acid sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identity with the amino acid sequence defined in 8E0 ΙΌ N0: 20, where this antibody interacts with ΙΡΝ-γ.

The invention also provides antibodies that can inhibit or modulate biological activity and / or specifically bind to ΙΡΝ-γ, where the heavy chain includes the amino acid sequence defined in 8E0 ΙΌ N0: 21, or its antigen-binding or immunologically functional immunoglobulin fragment, and the light chain includes the amino acid sequence defined in 8E0 ΙΌ N0: 33, or its antigen-binding or immunologically functional immunoglobulin fragment.

In some aspects of the invention, antibodies are provided that can inhibit or modulate biological activity and / or specifically bind to IΡN-γ containing a heavy chain and a light chain, where the heavy chain comprises a variable region of a heavy chain, and this variable region of a heavy chain includes a sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identity with the amino acid sequence defined in 8E0 ΙΌ N0: 21, and where the light chain includes the variable region of the light tse pi, wherein this variable region of the light chain comprises an amino acid sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identity with the amino acid sequence defined in 8E0 ΙΌ N0: 33, moreover, this antibody interacts with I -N-γ.

The invention also provides single chain antibodies, single chain Ρν antibodies, P (a b) antibodies, P (a b) 'antibodies and (Pa b') ₂ antibodies.

In particular aspects of the invention, there is provided a light chain comprising an amino acid sequence as defined in any of 8E0 ΙΌ N0: 8, 8E0 ΙΌ N0: 12, 8E0 ΙΌ N0: 16, 8E0 ΙΌ N0: 18, 8E0 ΙΌ N0: 20, 8E0 ΙΌ N0 : 22, 8Е0 ΙΌ N0: 31 or 8Е0 ΙΌ N0: 33, or its antigen-binding or immunologically functional immunoglobulin fragment.

In addition, the invention provides a heavy chain comprising an amino acid sequence as defined in any of 8E0 ΙΌ N0: 6, 8E0 ΙΌ N0: 10, 8E0 ΙΌ N0: 14, 8E0 ΙΌ N0: 17, 8E0 ΙΌ N0: 19, 8E0 ΙΌ N0: 21, 8Е0 ΙΌ N0: 30 or 8Е0 ΙΌ N0: 32, or its antigen-binding or immunologically functional immunoglobulin fragment.

The invention also relates to isolated human antibodies that specifically bind IΡN-γ, where such an antibody includes: (a) human heavy chain framework regions, complementarity determining the human heavy chain region 1 (CEC1 region), human heavy chain CEC2 region, and CEC3 - section of the human heavy chain; and (b) frame sections of the human light chain, 0ΌΒ1 region of the human light chain, C0K2 region of the human light chain and CEC3 region of the human light chain. In some aspects, the SOCL region of a human heavy chain may be a CEC1 region of a monoclonal antibody (mAb) heavy chain 1119, 1118, 1118 * or 1121, as shown in FIG. 12 and 8E0 ΙΌ N0: 34, and the CEC1 region of the human light chain may be the CEC1 region of the light chain of mAbs 1119, 1118, 1121 or 1121, as shown in FIG. 13 and 8E0 ΙΌ N0: 38, 8E0 ΙΌ N0: 39, or 8E0 ΙΌ N0: 40. In other aspects, the CEC2 region of the human heavy chain may be the CEC2 heavy chain region of mAb 1119, 1118, 1118 * or 1121, as shown in FIG. 12 and 8E0 ΙΌ N0: 35, and the C0K2 portion of the human light chain may be the CEC2 portion of the light chain of mAbs 1119, 1118, 1121 * or 1121, as shown in FIG. 13 and 8E0 ΙΌ N0: 41 or 8E0 ΙΌ N0: 42. In other aspects, the SHOC region of the human heavy chain is the CEC3 region of the heavy chain of mAbs 1119, 1118, 1118 * or 1121, as shown in FIG. 12 and 8E0 ΙΌ N0: 36 or 8E0 ΙΌ N0: 37, and the CEC3 region of the human light chain is the C0K3 region of the light chain of mAbs 1119, 1118, 1121 * or 1121, as shown in FIG. 13 and 8Е0 ΙΌ N0: 43 or 8Е0 ΙΌ N0: 44.

In addition, the invention provides methods of treating a disease associated with increased production or sensitivity to IΡN-γ and / or a disease mediated by ΓΡ ^ γ, comprising the step of administering to the needy a pharmaceutically effective amount of one or a plurality of monoclonal antibodies of the invention or its antigen binding or an immunologically functional immunoglobulin fragment.

The invention also provides methods for detecting the level of IΡN-γ in a biological sample, comprising the step of contacting the sample with the monoclonal antibody of the invention or an antigen binding fragment thereof.

The invention also provides an isolated antibody that can specifically bind to IΡN-γ and / or inhibit or modulate the biological activity of IΡN-γ and contains CEC3 more

- 4 011876 a free chain having an amino acid sequence consisting of: (a) an amino acid sequence consisting of at least 7 amino acids of 8EC GO N0: 36 in the same order and spatial arrangement as they are located in 8EC GO N0: 36; or (b) an amino acid sequence comprising 8E0 GO N0: 37. The antibody may further comprise a light chain CEB3 having an amino acid sequence comprising: (a) an amino acid sequence consisting of at least 8 amino acids 8E0 GO N0: 43 in the same order and spatial arrangement, as they are presented in 8E0 GO N0: 43; or (b) an amino acid sequence consisting of at least 9 amino acids of 8EC GO N0: 44 in the same order and spatial arrangement as they are shown in 8E0 GO N0: 44. The antibody may further comprise one or more SOY selected from the group consisting of 8EC EC GO N0: 34, 8EO GO N0: 35, 8EO GO N0: 38, 8EO GO N0: 40, 8EO GO N0: 40, 8EO GO N0: 41 and 8EO GO N0: 42. The SEV3 heavy chain may consist of at least amino acids 8E0 GO N0: 36, and SEY3 light chain may consist of at least amino acids 8E0 GO N0: 43.

In addition, the invention provides isolated antibodies that can specifically bind to the gel and / or inhibit or modulate the biological activity of the gel, containing C0H3 light chain having an amino acid sequence consisting of: (a) an amino acid sequence consisting of at least 8 amino acids 8E0 GO N0: 43 in the same order and spatial arrangement as they are located in 8E0 GO N0: 43; or (b) an amino acid sequence consisting of at least 9 amino acids 8E0 GO N0: 44 in the same order and spatial arrangement as they are located in 8E0 GO N0: 44. The antibody may further include a SOY having the amino acid sequence 8E0 GO N0: 35, 8Е0 GO N0: 38, 8ЕО GO N0: 39, 8ЕО GO N0: 40, 8ЕО GO N0: 41 or 8ЕО GO N0: 42.

An isolated antibody of the invention, which can specifically bind to the HE ^ y and / or inhibit or modulate the biological activity of the HE ^ y, may include 6 SOYs having at least amino acid sequences: (a) 8E0 GO N0: 34; (b) 8E0 GO N0: 35; (c) 8E0 GO N0: 36 or 8EO GO N0: 37; (d) 8EO GO N0: 38, 8EO GO N0: 39 or 8EO GO N0: 40; (e) 8EO GO N0: 41 or 8EO GO N0: 42 and (f) 8EO GO N0: 43 or 8EO GO N0: 44.

The isolated antibodies of the invention, which can specifically bind to HEU and / or inhibit or modulate the biological activity of HEU, can include an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or approximately 99% is identical to 8Е0 GO N0: 6, 8Е0 GO N0: 10, 8Е0 GO N0: 14 or 8Е0 GO N0: 30, and a comparison of the indicated amino acid sequence with 8E0 GO N0: 6, 8Е0 GO N0: 10, 8E0 GO N0: 14 or 8E0 GO N0: 30 encompasses at least 50 amino acids, and / or an amino acid sequence that is at least 80, 8 5, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% are identical to 8Е0 GO N0: 8, 8Е0 GO N0: 12, 8Е0 GO N0: 16 or 8Е0 GO N0: 31, and the comparison the specified amino acid sequence with 8E0 GO N0: 8, 8E0 GO N0: 12, 8E0 GO N0: 16 or 8E0 GO N0: 31 covers at least 50 amino acids.

In another aspect, antibodies of the invention, which can specifically bind to the GEC-y and / or inhibit or modulate the biological activity of the GEC-y, can include an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% is identical to 8E0 GO N0: 17, 8E0 GO N0: 19, 8E0 GO N0: 21 or 8E0 GO N0: 32, where the comparison of the specified amino acid sequence with 8E0 GO N0: 17, 8E0 GO N0: 19, 8E0 GO N0: 21 or 8E0 GO N0: 32 encompasses at least 50 amino acids, and / or an amino acid sequence that is at least n and 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% are identical to 8Е0 ГО N0: 18, 8Е0 ГО N0: 20, 8Е0 ГО N0: 22 or 8Е0 ГО N0: 33 where the comparison of the indicated amino acid sequence with 8E0 GO N0: 18, 8E0 GO N0: 20, 8E0 GO N0: 22 or 8E0 GO N0: 33 covers at least 50 amino acids. One amino acid sequence in these antibodies may include at least 5 amino acids 8E0 GO N0: 36 or 8E0 GO N0: 37 in the same order and spatial arrangement as they are located in 8E0 GO N0: 36 or 8E0 GO N0: 37, and / or one amino acid sequence in these antibodies may include at least 6 amino acids 8E0 GO N0: 43 or 8E0 GO N0: 44 in the same order and spatial arrangement as they are located in 8E0 GO N0: 43 or 8E0 GO N0: 44.

In one aspect of the invention, an antibody is provided that can be an isolated fully human antibody, where the antibody can inhibit or modulate the biological activity of a human HEU. In another aspect, the antibody of the invention, which may be an isolated fully human antibody, cannot inhibit or modulate the biological activity of the HEU monkeys and mice. In yet another aspect, the fully human antibody of the invention can inhibit or modulate the biological activity of human and chimpanzee chype, but it cannot inhibit or modulate the biological activity of the HEU monkey eupoto1-8 and / or mouse. In yet another aspect, replacing residue 19 in human GE-γ with aspartic acid and / or residue 20 with proline prevents inhibition or antagonizes

- 5 011876 effect in relation to the inhibition of the biological activity of human β-γ by this antibody. In addition, this antibody can inhibit the biological activity of the mutant variant of the ΙΡΝ-γ monkey super1to1di5, substituted at residues 19, 20 and 65 by histidine, serine and serine, respectively.

In other aspects, the isolated antibodies of the invention that can specifically bind to β-γ and / or inhibit or modulate the biological activity of β-γ may include an amino acid sequence of at least 80, 85, 90, 91, 92, 93, 94 , 95, 96, 97, 98, or approximately 99% identical to 8EC ΙΌ N0: 17, 8EC ΙΌ N0: 19, 8EC ΙΌ N0: 21 or 8EC ΙΌ N0: 32, and a comparison of the indicated amino acid sequence with 8EC ΙΌ N0: 17, 8EC ΙΌ N0: 19, 8EC ΙΌ N0: 21 or 8EC W N0: 32 covers at least 50 amino acids, and / or may include an amino acid sequence at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identical to 8EC ΙΌ N0: 18, 8EC ΙΌ N0: 20, 8EC ΙΌ N0: 22 or 8EC ΙΌ N0: 33, and the comparison of the specified amino acid sequence with 8EC ΙΌ N0: 18, 8EC ΙΌ N0: 20, 8EC ΙΌ N0: 22 or 8EC ΙΌ N0: 33 covers at least 50 amino acids.

In yet another embodiment, the invention encompasses an isolated antibody that can specifically bind to NI ^, comprising: (a) an amino acid sequence comprising 8EC ΙΌ N0: 6, 8EC ΙΌ N0: 10, 8EC ΙΌ N0: 14 or 8EC ΙΌ N0: 30, or a fragment of one of these sequences and (b) an amino acid sequence including 8EC ΙΌ N0: 8, 8EC ΙΌ N0: 12, 8EC ΙΌ N0: 16 or 8EC ΙΌ N0: 31, or a fragment of one of these sequences. An antibody may contain a heavy chain and a light chain. An antibody may contain: (a) 8EC ΙΌ N0: 6, 8EC ΙΌ N0: 10, 8EC ΙΌ N0: 14 or 8ЕО ΙΌ N0: 30 and (b) 8EC ΙΌ N0: 8, 8ЕО ΙΌ N0: 12, 8ЕО ΙΌ N0: 16 or 8ЕО ΙΌ N0: 31.

In addition, any of the antibodies of the invention may be humanized antibodies or fully human antibodies.

The invention also provides polynucleotides, including isolated polynucleotides, which encode any of the antibodies of the invention or portions thereof, as described herein, including SER sections, variable regions of the heavy chain, variable regions of the light chain, single chain antibodies, single chain Ρν antibodies, P (a b) -antibodies, P (ab) '- antibodies and (PaB') ₂ -antibodies. In addition, the invention provides vectors containing such polynucleotides and host cells, possibly mammalian cell hosts, containing such polynucleotides and / or vectors. Antibodies of the invention can be obtained by culturing such host cells.

Specific preferred embodiments of the invention will become apparent from the following more detailed description of some preferred embodiments and claims.

A brief description of the graphic materials

In FIG. 1A-1B depicts the nucleotide sequence of a cDNA region (Fig. 1A; 8EC ΙΌ N0: 1) encoding the amino acid sequence (Fig. 1B; 8EC ΙΌ N0: 2) of the constant region of the heavy chain of anti-CW ^ antibodies 1118, 1118 *, 1119, 1121 and 1121 *.

In FIG. 2A-2B shows the nucleotide sequence of the cDNA region (Fig. 2A; 8EC ΙΌ N0: 3) encoding the amino acid sequence (Fig. 2B; 8EC Ш N0: 4) of the light chain constant region of the anti-SN ^ antibodies 1118, 1118 *, 1119, 1121 and 1121 *.

In FIG. 3A-3B depicts the nucleotide sequence of a cDNA region (FIG. 3A; 8EC C N0: 5) encoding the amino acid sequence (FIG. 3B; 8EC C ΙΌ N0: 6) of the variable region of the heavy chain of anti-CMM ^ antibody 1119.

In FIG. 4A-4B shows the nucleotide sequence of a cDNA region (Fig. 4A; 8EC ΙΌ N0: 7) encoding the amino acid sequence (Fig. 4B; 8EC ΙΌ N0: 8) of the light chain variable region of anti-GBA antibody 1119.

In FIG. 5A-5B show the nucleotide sequence of a cDNA region (FIG. 5A; 8EC ΙΌ N0: 9) encoding the amino acid sequence (FIG. 5B; 8EC Ш N0: 10) of the heavy chain variable region of anti-IΡN-γ antibody 1118. FIG. 5C shows the amino acid sequence of the variable region of the heavy chain (8EC W N0: 30) of the anti-IΡN-γ antibody 1118 *. In FIG. 5Ό presents the nucleotide sequence of the variable region of the heavy chain (8EC ΙΌ N0: 56) anti-HSC ^ antibodies 1118 *.

In FIG. 6A-6B show the nucleotide sequence of a cDNA region (Fig. 6A; 8EC C N0: 11) encoding the amino acid sequence (Fig. 6B; 8EC ΙΌ N0: 12) of the light chain variable region of the anti-IΡN-γ antibody 1118 or 1118 *.

In FIG. 7A-7B show the nucleotide sequence of a cDNA region (Fig. 7A; 8EC ΙΌ N0: 13) encoding the amino acid sequence (Fig. 7B; 8EC Ш N0: 14) of the variable region of the heavy chain of an anti-IΡN-γ antibody 1121 or 1121 *.

In FIG. 8A-B presented the nucleotide sequence of the cDNA region (FIG. 8A; 8EC ΙΌ N0: 15) encoding the amino acid sequence (FIG. 8B; 8EC ΙΌ N0: 16) of the light chain variable region of the anti-IΡN-γ antibody 1121. FIG. 8C shows the amino acid sequence (8EC ΙΌ N0: 31) of the light chain variable region of the anti-IΡN-γ antibody 1121 *. In FIG. 8Ό shows the nucleotide sequence (8EC ΙΌ N0: 57) of the light chain variable region of the anti-IΡN-γ antibody 1121 *.

FIG. 9 contains a graph showing the neutralization or inhibition of biological

- 6 011876 ΙΡΝ-γ activity in A549 bioanalysis with monoclonal antibodies 1119, 1118 and 1121.

FIG. 10 contains a graph showing the neutralization or inhibition of the biological activity of β-γ in the THP-1 / HLA EV bioanalysis with monoclonal antibodies 1119, 1118 and 1121.

FIG. 11 contains a graph demonstrating the neutralization or inhibition of the biological activity of ΙΡΝ-γ in the bioassay of whole blood (two donors) by monoclonal antibody 1119.

FIG. 12 shows a comparison of the amino-terminal region (including the variable region) of the anti-ΙΡΝ-γ monoclonal antibody heavy chains, designated 1118, 1118 *, 1121 and 1119. These sequences include the signal sequence encoded by the cDNA isolated in Example 3. The signal sequence extends from position 1 to 19. The CMEA sections are underlined. As shown in this figure, SIV1 covers amino acids 50-54, SIV2 covers amino acids 69-85 and SIV3 covers amino acids 118-125. Numbering according to the system according to KaLa1 e1 a1. (1991, 8cc | expss5 o £ Prgo1ss8o £ 1tipo1ofsa1 I1e1eek1. Riebie Nea11e 8tu1e Ν.Ι.Η., VeShekba, ΜΌ) begins with the first amino acid of the mature antibody and excludes the signal sequence. Thus, position 20 in this figure will correspond to position 1 in KaLa1 e1 a1. (see above).

FIG. 13 shows a comparison of the amino-terminal portion (including the variable region) of the light chains of anti-β-γ monoclonal antibodies designated 1118, 1121, 1121 * and 1119. The sequences include the signal sequence encoded by the cDNA isolated in Example 3. The signal sequence extends from position 1 to 20. SIW areas are underlined. As noted in this figure, SIV1 covers amino acids 44-55, SIV2 covers amino acids 71-77 and SIV3 covers amino acids 110-118. Since the numbering system is KaLa1 e1 a1. (above) excludes the signal sequence, then position 21 in this figure corresponds to position 1 in KaLa1 e1 a1.

FIG. 14 shows the production of ΙΡ-10 in response to ΙΡΝ-γ with whole blood taken from chimpanzees for 2 or 1 week (s) (lines indicated by -2 and -1 in Fig. 14) before or after 2, 8, 15, 29 or 36 days (lines indicated by 2, 8, 15, 29 or 36 in Fig. 14) after the start of a course consisting of 3 injections of anti-ΙΡΝ-γ antibody, which was given 1 time per week.

FIG. 15 is similar to FIG. 14, except that they used blood from another chimpanzee.

DETAILED DESCRIPTION OF THE INVENTION

The section headings used herein are for organizational purposes only and should not be interpreted as limiting the subject matter described. All references cited in this application are expressly incorporated herein by reference for any purpose.

Definitions

Standard techniques can be used for the synthesis of recombinant DNA, oligonucleotides, and for tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification procedures can be carried out according to the manufacturer's instructions or performed as usual in the art or as described herein. The aforementioned techniques and procedures can be generally embodied by standard methods well known in the art and described in various general and more specific references that are cited and discussed in the present description. See, for example, 8attook e1 a1., 2001, MOTESIАВV ί'ΤΘΝΙΝΟ: BAVOVATOV ΜΑΝυΑφ 36 eb., Co1b 8rppd Nagyog baogoga Fts88, Co16 8rppd Nagog, Ν.Υ., which is incorporated here by reference for any purpose. If specific definitions are not given, then the nomenclature and laboratory procedures used in this connection, as well as the methods of analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described here, are well known and commonly used in this field. Standard techniques can be used for chemical synthesis, chemical analyzes, manufacture and delivery of a pharmaceutical product, and patient care.

Unless otherwise indicated, the following terms used in the present description, should be understood as having the following meanings.

The term “isolated polynucleotide” as used herein should mean a polynucleotide of genomic, cDNA or synthetic origin, or some combination thereof, whereby the isolated polynucleotide is: ) is associated with a polynucleotide with which it is not associated in nature, or (3) does not occur in nature as part of a longer sequence.

The term “isolated protein” as used herein means that a given protein: (1) is free from at least some other proteins with which it is usually found in nature, (2) is essentially free from other proteins from the same source, for example from of the same species, (3) is expressed by a cell of another species, (4) has been separated from at least about 50% of polynucleotides, lipids, carbohydrates or other substances with which it is associated in nature, (5) is not associated (by covalent or non-covalent interactions) with the protein sites with which this isolated a protein is bound in nature, (6) is functionally bound (by covalent or non-covalent interaction) with a polypeptide with which it is not bound in nature, or (7) is not found in nature. Such an isolated protein may be encoded by genomic DNA, cDNA, mRNA or other synthetic RNA

- 7 011876 or any combination thereof. Preferably, the isolated protein is essentially free of proteins or polypeptides or other impurities that are found in its natural environment, which usually interferes with its use (therapeutic, diagnostic, preventive, research, or some other).

The terms polypeptide or protein means one or more than one chain of amino acids, where each chain includes amino acids covalently linked by peptide bonds, and where the specified polypeptide or protein can contain many chains non-covalently and / or covalently linked together by peptide bonds having a sequence of native proteins , i.e. proteins produced by natural and, in particular, non-recombinant cells or genetically engineered or recombinant cells, and contain molecules having the amino acid sequence of the native protein, or molecules having deletions, insertions and / or substitutions of one or more amino acids of the native sequence. The terms polypeptide and protein, in particular, encompass anti-ΙΡΝ-γ antibodies or sequences that have deletions, insertions and / or substitutions of one or more amino acids of the anti-ΙΡΝ-γ antibody. Thus, a polypeptide or protein may include one (called a monomer) or multiple (called a multimer) amino acid chains.

The term “polypeptide fragment” refers to a polypeptide, which may be monomeric or multimeric, that has an amino terminal deletion, a carboxy terminal deletion and / or an internal deletion, or a replacement for a natural or recombinantly produced polypeptide. In some embodiments, the polypeptide fragment may include an amino acid chain of at least 5 to about 500 amino acids in length. It is understood that in some embodiments, the fragments have at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length. Particularly useful polypeptide fragments contain functional domains, including binding domains. In the case of anti-ΙΡΝ-γ antibodies, useful fragments include, but are not limited to, the SEA site, especially the SEA3 site of the heavy or light chain; heavy or light chain variable domain; a portion of the chain of an antibody or precisely its variable region, including two SOY; etc.

As used herein, the term “immunologically functional immunoglobulin fragment” refers to a polypeptide fragment that at least contains CIA regions of the immunoglobulin heavy and light chains. An immunologically functional immunoglobulin fragment of the invention is capable of binding to an antigen. In preferred embodiments, the antigen is a ligand that specifically binds to a receptor. In these embodiments, the binding of the immunologically functional immunoglobulin fragment of the invention prevents or inhibits the binding of the ligand to its receptor, interrupting the biological response resulting from the binding of the ligand to the receptor. Preferably, the immunologically functional immunoglobulin fragment of the invention specifically binds to β-γ. Most preferably, this fragment specifically binds and / or inhibits or modulates the biological activity of human β-γ.

As used herein, the term natural refers to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in the body (including viruses), which can be isolated from a natural source and not specifically modified by humans, is natural.

The term functionally related means that the components to which this term is applied are related to each other so that they can perform their inherent functions under suitable conditions. For example, a transcriptional control sequence operably linked to a protein coding sequence is ligated to the latter so that, under conditions compatible with the transcriptional activity of the control sequences, expression of the protein coding sequence is achieved.

As used herein, the term “control sequence” refers to a polynucleotide sequence that can affect the expression, processing or intracellular localization of coding sequences with which they are ligated. The nature of such control sequences may depend on the host organism. In specific embodiments, prokaryotic transcription control sequences may include a promoter, a ribosome binding site, and a transcription termination sequence. In other specific embodiments, sequences that control transcription in eukaryotes may contain promoters that include one or many recognition sites for transcription factors; sequences enhancing transcription; transcription termination sequences and polyadenylation sequences. In some embodiments, the control sequences may include leader sequences and / or fusion partner sequences.

The term polynucleotide as referred to herein means single or double stranded nucleotide polymers of at least 10 bases in length. In some embodiments, the nucleotides comprising the polynucleotide may be ribonucleotides or deoxyribonucleotides or a modified form of any type of nucleotide. These modifications include base modifications, such as bromuridine, ribose modifications, such as arabinoside and 2 ', 3'-dideoxy

- 8 011876 bose, and modifications of internucleotide bonds, for example phosphorothioate, phosphorodithioate, phosphorus selenate, phosphorodislenoate, phosphoroanilothioate, phosphoranilate and phosphoroamidate. The term polynucleotide, in particular, includes single and double stranded forms of DNA.

The term oligonucleotide as referred to herein includes natural and modified nucleotides linked together by natural and / or non-naturally occurring oligonucleotide bonds. Oligonucleotides are a subset of polynucleotides that are usually single-stranded and have a length of 200 bases or less. In some embodiments, the oligonucleotides are 10-60 bases in length. In some embodiments, the oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, or 20-40 bases in length. Oligonucleotides can be single-stranded or double-stranded, for example, for use in the construction of a gene mutant. The oligonucleotides of the invention may be sense or antisense oligonucleotides with respect to the protein coding sequence.

Unless otherwise indicated, the left end of the single-stranded polynucleotide sequence is the 5'-end; the direction of the double-stranded polynucleotide sequence to the left is called the 5'-direction. The direction 5'-3 'of the addition of emerging RNA transcripts is called the direction of transcription; regions of the DNA chain sequence having the same sequence as RNA and which are located 5 ′ from the 5 ′ end of the RNA transcript are denoted as sequences in the opposite direction; regions of a DNA chain sequence having the same sequence as RNA and located 3 ′ from the 3 ′ end of the RNA transcript are referred to as forward sequences.

The term natural nucleotides includes deoxyribonucleotides and ribonucleotides. The term modified nucleotides includes nucleotides with modified or substituted sugar groups and the like. The term oligonucleotide bonds includes oligonucleotide bonds such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilate, phosphoroamidate and the like. See, for example, Laplaxe e! A1., 1986, Νιιοί. As1bk Vek., 14: 9081; 81ec e! A1., 1984, 1. At. Sb. 8oc., 106: 6077; 81esh e! A1., 1988, No. c1. As1bk Vek., 16: 3209; Ζοη e! A1., 1991, Apb-Sapseg Tsgcd Oekschp. 6: 539; Ζοη e! a1., 1991, ΟΕΙΟΟΝ ^ ΕΕΟΤΙΌΕδ ΑΝΏ ΑΝΑΕΟΟυΕδ: A RABSAΑ ΑРРВΟΑСΗ, pp. 87-108 (E. Eskk! Esh, Εά.), Οχ & τά ishuetkyu Rgekk, Οχ & τά Епд1апб; §! Ec e! A1., patent υδ 5151510; ib1tapp apb Reutap, 1990, Sbetka1 Beu1e ^ k, 90: 543, the contents of which are hereby incorporated by reference for any purpose. The oligonucleotide may include a detectable label to facilitate detection of the oligonucleotide or its hybridization.

The term vector is used to refer to any molecule (eg, nucleic acid, plasmid or virus) used to transfer encoded information to a host cell.

The term expression vector refers to a vector that is suitable for transformation of the host cell and contains nucleotide sequences that direct and / or control the expression of the embedded heterologous nucleotide sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing if introns are present.

The term host cell is used to refer to a cell into which a nucleotide sequence has been introduced or can be introduced, and which then expresses or is capable of expressing a selected gene of interest. This term includes the progeny of the parent cell, regardless of whether the progeny is identical in morphology or genetic structure to the original parent or not, as long as the selected gene is present.

The term transduction is used to refer to gene transfer from one bacterium to another, usually using phage. Transduction also refers to the capture and transfer of eukaryotic cell sequences by retroviruses.

The term transfection is used to mean the capture of foreign or exogenous DNA by a cell, and the cell is transfected when exogenous DNA has been introduced under the cell membrane. A number of transfection methods are well known in the art and are disclosed here. See, for example, Otabat e! A1., 1973, Ulodod 52: 456; 8atto eok! a1., 2001, ΜΟ ^ ΕСυ ^ ΑВ С ^ ΟNIN6, ^ ΑΒΟВΑΤΟВΥ МАА'Сак. So1b 8rppd Nagbot Laibota! Eamk e! A1., 1986, BA81C ΜΕΤΗΟΌδ ΙΝ ΜΟ ^ ΕСυ ^ ΑВ ΒΙΟ6ΟΟΥ, Ε1 ^ \ ^ γ; and Sby e! A1., 1981, Oepe 13: 197. Such methods can be used to introduce one or more exogenous DNA groups into suitable host cells.

As used herein, the term “transformation” refers to a change in the genetic characteristics of a cell, and the cell is transformed when it has been modified to contain new DNA. For example, a cell is transformed when it is genetically modified compared to its native state. After transfection or transduction, the DNA transformant can recombine with the DNA of the cell through physical integration into the chromosome of the cell, or it can be maintained temporarily as an episomal element without replication, or it can replicate independently as a plasmid. A cell is considered stably transformed when such DNA is replicated simultaneously with cell division.

The term natural or native, when used in relation to biological substances, such as nucleic acid molecules, polypeptides, host cells and the like, refers to substances that are found in nature and not subjected to manipulation by humans. Similarly

- 9 011876 as used herein, the term non-natural or non-native refers to a substance that is not found in nature or that has been structurally modified or synthesized by humans.

The term “antigen” refers to a molecule or region of a molecule capable of binding to a selectively binding agent such as an antibody, and further such that can be used by an animal to produce antibodies capable of binding to an epitope of that antigen. An antigen may have one or more epitopes.

The term epitope includes any determinant, preferably a polypeptide determinant, capable of specifically binding to an immunoglobulin or T cell receptor. An epitope is a region of antigen that binds to an antibody. In some embodiments, epitope determinants contain chemically active surface moieties of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl, and in some embodiments may have specific three-dimensional structural characteristics and / or specific charge characteristics. In some embodiments, the antibody is said to specifically bind an antigen, preferably recognizing its target antigen in a complex mixture of proteins and / or macromolecules. The antibody is said to specifically bind antigen when the equilibrium dissociation constant is not more than 10 ^-7 or 10 ^-8 M. In some embodiments, the equilibrium dissociation constant can be no more than 10 ^-9 or no more than 10 ^-10 M.

As used here, if the first sequence consists of, for example, 10 amino acids of the sequence ΡΑ8Ο8ν888Υ (8Е0 GO N0: 56), then the other sequence has 7 amino acids in the same order and spatial arrangement as they occur in the first sequence, if 7 amino acids are identical to amino acids in this sequence and occur in the same positions relative to each other as they occur in this sequence. For example, the sequence ΚΑΑΑΑνδδδΥ (8Е0 ГО N0: 57) has 7 amino acids in the same order and spatial arrangement as they are found in ΚΑ§Ο§ν888Υ (8Е0 ΙΌ N0: 56). However, this is not true for the sequence ΚΑδδνδδδΥ (8Е0 ГО N0: 58), since it contains an internal deletion compared to ΚΑδΟδνδδδΥ (8Е0 ΙΌ N0: 56), where 3 and 6 amino acids are on the sides of the deletion. Thus, it has no more than 6 amino acids in the same order and spatial arrangement as the first sequence. The shortest possible sequence, which can have 7 amino acids in the same order and spatial arrangement as in ΡΑ8Ο8ν888Υ (8Е0 GO N0: 56), will have 7 amino acids in length, for example 8Ο8ν888 (8Е0 GO N0: 59).

The term identity, known in this field, refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, which is determined by comparing their sequences. Depending on the circumstances, in this area, identity also means the degree of affinity between nucleic acid molecules or polypeptides, which is determined by the coincidence of the chains of two or more nucleotide or two or more amino acid sequences. Identity characterizes the percentage of identical matches between the smaller of two or more sequences with cuts for matching (gift idptep! H) (if any) investigated using a specific mathematical model or computer program (i.e. algorithms).

The term similarity is used in the art in relation to a related concept, but, unlike identity, similarity refers to a characteristic of kinship, including both identical matches and conservative substitution matches. If two polypeptide sequences have, for example, 10/20 identical amino acids and the remainder is all non-conservative substitutions, then the percent identity and similarity will be 50%. If in the same example there are five additional provisions where conservative substitutions exist, then the percent identity remains 50%, and the percentage similarity will be 75% (15/20). Thus, in cases where there are conservative substitutions, the percentage similarity between the two polypeptides will be higher than the percentage identity between the two polypeptides.

The identity and similarity of related nucleic acids and polypeptides can be easily determined by known methods. Such methods include, but are not limited to, the methods described in S0ΜPυΤLΤI0NЛ ^ ХУЕЕСЕЕЛН ΒΙ0Ε00Υ (Езк, Α.Μ., еб.), 1988, 0x1otb E1i / ezp \ Prziez, Hei Wagk: B10C0MPiTi8Ti6: 0. 1EST8 (8th, Ό.№., Eb.), 1993, Lsas1e11is Rgezz, Hei ¥ o1k; S0MRITEV LNL ^ Υ8I8 0Е ΌΑΤΑ, Paradise 1 (ΟτΟη, Α.Μ., appb Οτίίίϊη, N.O., ebz.),

1994, Nitapa Rgezz, Hei 1etzeu, νοη Neide, O., .- ^ .- ^^ 818 W M0EESELN ΒΙ0Ε00Υ,

1987, Lsabeiis Rgesz; 8Е ^ υЕNСЕ ΑNΑ ^ Υ8I8 PK1MEK. (Olzkot, M. apb Oethegeih, 1., ebz.), 1991, M. 8! Osc! Op Prgezz, He SATSHO e! A1., 1988, 8 ILΜ 1. ρρρ ^ 6 Mach., 48: 1073; and ityp e! A1., 1998, ΒΙ0Ε00Ι (ΑΕ 8Е ^ υЕNСЕ ЛНЛ ^ Υ8I8, Сатппде Е4и \ еззП \ Ргзз.

Preferred methods for determining identity are designed to give the greatest match between the sequences in question. Methods for determining identity are described in publicly available computer programs. Preferred methods using computer programs to determine the identity between the two sequences include, but are not limited to, the CCA software package, including 6LR (Eetegeih e! A1., 1984, N ^ 1. 6α6. Vez., 12: 387;

- 10 011876

Sepebsk Sotrieg Sgoir, Leningrad OG Χνίδοοηδίη. Mabcop, XVI). VABTR, VABTH and EABTA (AXII 1 e1 a1., 1990, 1. Mo1. Vu1., 215: 403-410). The VABTX program can be obtained at the National Center for Biotechnological Information (VLB1) and from other sources (Vlabt Mapia1. A1 (ksNi1 e1 a1. ΝίΈ / ΝΕ-Μ / ΝΙΗ VeShekba. MI 20894; A11xNi1 e1 a1., 1990, above). The Smith-Waterman algorithm (Btw & Vaΐe ^ tai) can also be used to determine identity.

Some matching schemes for comparing two amino acid or polynucleotide sequences can only match a short portion of these two sequences, and this small comparable portion can have very high sequence identity, even if there is no significant relationship between the two full-length sequences indicated. Accordingly, in some embodiments, the selected matching method (CAP program) will give a comparison encompassing at least 50 consecutive amino acids of the target polypeptide. In some embodiments, the comparison may encompass at least 60, 70, 80, 90, 100, 110, or 120 amino acids of the target polypeptide. If the polynucleotides are compared using CAP, the comparison may cover at least about 100, 150 or 200 nucleotides, which may be sequentially arranged.

For example, when using the computer-assisted CAP algorithm (Sepebsk Sotrieg Sgoir, Lpkegiu OG Χν ^^ ηκ ^ 'ΐ, Mab1cop, VI), two polypeptides for which it is necessary to determine the percent identity of the sequences are combined with the optimal coincidence of their corresponding amino acids (match coverage (talis ) defined by this algorithm). In some embodiments, the penalty for introducing a space (the Orespd repaIu gift) (which is calculated as the triple average diagonal; where the average diagonal is the average diagonal of the used comparison matrix; the diagonal is the score or number assigned to each exact amino acid match by a specific comparison matrix) and a penalty for continuing the gap (gift ex! epcup repaIu) (which usually amounts to one tenth of the penalty for making a deletion), as well as a comparison matrix such as RAM 250 or LOB 62, and Use in conjunction with the algorithm. In some embodiments, this algorithm also uses a standard comparison matrix (see LauIoGG e1 a1., 1978, Abak oG Prgoesh Beschepse apb 8bis1ige, 5: 345-352 for the comparison matrix RAM 250; NetkoGG e1 a1., 1992, Proc. Asab. Asab Bsk I8A, 89: 10915-10919 for the comparison matrix VOBIM 62).

In some embodiments, parameters for comparing polypeptide sequences include the following:

algorithm: Yeeb1etap e1 a1., 1970, I. Mo1. Vu1., 48: 443-453;

comparison matrix (Sotrapkop tabgh): VOBOBIM 62 from NeshkoGG e1 a1., 1992, above;

fine for introducing a gap (Sar RepaIu): 12;

Penalty on continuation of the gap (Сар бепд1И RepaIu): 4;

threshold of similarity (Tigekio1b oG Bshbagyu): 0.

CAP program can be used with the above parameters. For nucleotide sequences, the parameters may include a penalty for introducing a space of 50 and a penalty for continuing a space of 3, i.e. a penalty of 3 for each character in each space. In some embodiments, the aforementioned parameters are default parameters for comparisons of polypeptides (together with no penalty for trailing spaces) using the CAP algorithm.

As used here, 20 standard amino acids and their abbreviations have standard use. See 1MMIOJOS ¥ - A BUKTNEB1B, 2pb Ebüyup, (E.B.So1iB apb Ό.Κ .. Step, Ebk.), Bshayeg Akkos1a1ek: Bipbebapb, MA, 1991, is incorporated here by reference for any purpose. Stereoisomers (e.g. Ό-amino acids) 20 standard amino acids; unnatural amino acids, such as α-, α-disubstituted amino acids, Ν-alkyl amino acids, lactic acid and other non-standard amino acids, may also be suitable components of the polypeptides of the invention. Examples of non-standard amino acids include 4-hydroxyproline, γ-carboxyglutamate, ε-Ν, Ν, Ν-trimethyllysine, εΝ-acetylysine, O-phosphoserine, Ν-acetylserin, Ν-formylmethionine, 3-methylhistidine, 5-hydroxylisine, σ-Ν- methylarginine and other similar amino acids and imino acids (e.g. 4-hydroxyproline). In the designation of polypeptides used here, the left direction is the amino terminal direction, and the right direction is the carboxy terminal direction in accordance with standard application and arrangement.

Natural residues can be divided into classes based on the common properties of the side chains:

1) hydrophobic: norleucine (Hoog), Me1, A1a, Wa1, Lei, 11e;

2) neutral hydrophilic: Suk, Running, TIG, Akp, C1p;

3) acid: Acre, C1i;

4) the main ones: H1k, Luk, Agd;

5) residues that affect the orientation of the chain: C1u, Rgo; and

6) aromatic: Tgr, Tug, RIE.

Conservative amino acid substitutions may mean replacing one representative of one of these classes with another representative of the same class. Conservative Amino Acid Substitutions

- 11 011876 may mean unnatural amino acid residues, which are usually included in the chemical synthesis of peptides, and not as a result of synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid groups.

Non-conservative substitutions may mean replacing a representative of one of these classes with a representative from another class. Such substituted residues can be introduced into regions of the human body that are homologous to non-human antibodies, or into non-homologous regions of the molecule.

When creating such substitutions in accordance with specific embodiments, the hydropathic amino acid index may be taken into account. Each amino acid was assigned a hydropathic index based on its hydrophobicity and charge characteristics. The indices are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9) and arginine (-4.5).

In this area, the importance of the hydropathic amino acid index lies in the assignment of an interactive biological function to a protein (see, for example, Ku1c s1 a1., 1982, 1. Μοί. ΒίοΙ. 157: 105-131). It is known that some amino acids can be replaced by other amino acids having a similar hydropathic index or score, while maintaining a similar biological activity. When creating substitutions based on the hydropathic index, in some embodiments, there is a substitution of amino acids whose hydropathic indices are within ± 2. In some embodiments, substitutions are included with indices that are within ± 1, and in some embodiments, substitutions with indices that are within ± 0.5 are included.

It is also understood in the art that replacement of similar amino acids can be efficiently done based on hydrophilicity, especially if the biologically functional protein or peptide created in this way is intended for use in immunological embodiments as disclosed herein. In some embodiments, the largest local average hydrophilicity of a protein, determined by the hydrophilicity of its neighboring amino acids, correlates with its immunogenicity and antigenicity, i.e. with the biological property of this protein.

The following hydrophilicity values were assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+ 3.0 ± 1); glutamate (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). When creating substitutions based on similar hydrophilicity values, in some embodiments, amino acid replacement is included whose hydrophilicity values are within ± 2, in some embodiments hydrophilicity values that are within ± 1, and in some embodiments hydrophilicity values that are within within ± 0.5. Based on hydrophilicity, epitopes from primary amino acid sequences can also be identified. These sites are also designated as epitope crustal sites.

Exemplary amino acid substitutions are presented in table. one.

Table 1

Amino Acid Substitutions

Original Residues Replacement Examples Preferred replacements A1a Wa1, Lei, Not Ya1 Ag® Bk, <e1n, azp Bz

Original Residues Sample replacements Preferred replacements Azp C1p C1p Azr C1 and O1i Suz Zeg, A1a Zeg O1p Azp Azp 61and Azr Azr C1u Rgo, A1a A1a ΗΪ3 Azp, O1n, bk, Agd Agv Not Ley, Ya1, Μοί, A1a, Pye, Norleucine Bie Bie Norleucine, Pe, Va1, Me (, A1a, Pb Pe Bz Arg, 1,4-Diamino butyric acid, O1p, Alp Ag§ Me! Lye, Lye, Pe Bie Pb Ley, Wah !, Pe, A1a, Tug Bie Rgo A1a O1u Zeg Ty, A1a, Suz Thr Thr Zeg 8eg Tgr Here, pb Here Tug Thr, Pb, Thb, Zeg Pb Ya1 Not, Meg, Ley, Lye, A1a, Norleucin Bie

- 12 011876

The practitioner will be able to determine suitable variants of the polypeptide described herein using well-known methods. In some embodiments, one of skill in the art can identify suitable regions of a molecule that can be altered without disturbing activity by target regions that are not believed to be significant to the activity. In other embodiments, one of skill in the art can identify residues and regions of molecules that are conserved among similar polypeptides. In other embodiments, even regions that may be essential for biological activity or for structure may be subjected to conservative amino acid substitutions without impairing biological activity or without undesired effect on the polypeptide structure.

In addition, a person skilled in the art can analyze the results of structural-functional studies identifying residues in similar polypeptides that are important for activity or structure. Given this comparison, one skilled in the art can predict the significance of amino acid residues in a protein that correspond to amino acid residues that are important for activity or structure in similar proteins. One of skill in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

One of skill in the art can also analyze the three-dimensional structure and amino acid sequence relative to that in similar polypeptides. Given such information, one skilled in the art can predict the coverage of a comparison of the amino acid residues of an antibody with respect to its three-dimensional structure. In some embodiments, one of ordinary skill in the art may prefer not to make radical changes to the amino acid residues that are thought to be on the surface of this protein, since such residues may be involved in important interactions with other molecules. Moreover, a person skilled in the art can prepare test variants containing a single amino acid substitution in each desired amino acid residue. These options can then be analyzed using activity assays known to those skilled in the art. Such options can be used to collect information about suitable options. For example, if it is found that a change in a specific amino acid residue has resulted in impaired, undesirably reduced or inappropriate activity, then variants with such a change can be avoided. In other words, based on the information gathered from such routine experiments, one skilled in the art can easily determine amino acids in which additional substitutions should be avoided, either single or in combination with other mutations.

A number of scientific publications have been devoted to the prediction of secondary structure. See, for example, My1 !, 1996, Pt. Op. ίη Vu! Yes. 7: 422-427; Failures e! A1., 1974, Vusyesh151gu 13: 222-245; Failures e! A1., 1974, Vyushiesh15yu 113: 211-222; Failures e! A1., 1978, Αάν. Ephuto 1. Pe1a !. Lgea ₈ Mo1. Vu1. 47: 45-148; Failures e! A1., 1979, App. Ret. Vusyesh. 47: 251-276; and crashes e! A1., 1979, Vyuru ₈ . 1.26: 367-384. Moreover, computer programs are currently available to help predict the secondary structure. One way to predict secondary structure is based on homology modeling. For example, two polypeptides or proteins that have a sequence identity of more than 30% or similarity of more than 40% often have similar structural configurations.

Modern database growth of the structure of proteins (REV, pro! Em 51gis! Iga1 ba! E aba ₈₎ provided enhanced predictability of secondary structure, including the potential number of folds within a polypeptide or protein structure. See Butt! A1., 1999, Yis1. Ac1b. Ke5. 27: 244-247. It was suggested (Hegepeg e! A1., 1997, Sigg. Or. 81gis !. Vu1. 7: 369-376) that there is a limited number of folds in a given polypeptide or protein and that if a critical number of structures are allowed, the structural prediction becomes much more accurate.

Additional methods for predicting the secondary structure include enumeration (! Igeabsd) (1op ₈ , 1997, Sigg. Orsh. 8! Gis !. Vu1. 7: 377-87; 81rp1 e! A1., 1996, 8! Gis! Yee 4: 15 -19), profile analysis (Bo \\! E e! A1., 1991, 8c1epse 253: 164-170; Cb ^ cat e! A1., 1990, Me111. Ephut. 183: 146-159; Cb ^ cat e ! a1., 1987, Prgos. Ba! Asab. 8cg 84: 4355-4358) and evolutionary connection (see Ho1t, 1999, above; and Wgeppeg, 1997, above).

According to some embodiments, amino acid substitutions are substitutions that: (1) decrease the sensitivity to proteolysis, (2) reduce the sensitivity to oxidation, (3) change the binding affinity of the resulting protein complexes, (4) change the binding affinities and / or (5) provide or modify other physicochemical or functional properties of such polypeptides. According to a particular embodiment, single or multiple amino acid substitutions (in some embodiments, conservative amino acid substitutions) can be made in the natural sequence (in some embodiments, in the region of the polypeptide outside the domain (s) forming the intermolecular contacts). In preferred embodiments, a conservative amino acid substitution usually does not substantially change the structural characteristics of the related sequence (for example, amino acid substitution should not disrupt the helix that is in the related sequence or disrupt other types of secondary structure that characterizes the related sequence). Examples of secondary and tertiary structures of polypeptides adopted in this field of technology are described in RVOTEB8, 8TvISTIvE8 Ь MOBILE ΡΗΙΝ (ΊΡ1.Ε8. (Ίϊαμίιίοιι...), 1984, No. N. Egeetap apy Sotrapu, WogΤΙΘΝ;

TO ΡΚΘΤΕΙΝ 8TKiSTiKE (S. Vgapyep apy 1. Toohe. Nek.), 1991, Oag1apy RiSHypd, Νονν Wagk, Ν.Υ .; and T1yugp1op e1 a1., 1991, No. Iige 354: 105, each of which is incorporated herein by reference.

Peptide analogues are commonly used in the pharmaceutical industry as non-peptide drugs with properties similar to those of a model peptide. These types of non-peptide compounds are called peptide mimetics or peptidomimetics. See Eaisier, 1986, Lyu. Aegis Century. 15: 29; Yeeb & Ege1ishdeg, 1985, ΉΝ8, p. 392; and Esapk e1 a1., 1987, 1. May. Eat. 30: 1229, which are incorporated herein by reference for any purpose. Such compounds are often created by computer molecular modeling. Peptidomimetics that are structurally similar to therapeutically useful peptides can be used to obtain a similar therapeutic or prophylactic effect. Typically, peptidomimetics are structurally similar to the parent polypeptide (i.e., a polypeptide that has a biochemical property or pharmacological activity), such as a human antibody, but has one or more peptide bonds, possibly replaced by a bond selected from —CH ₂ —IN—, - CH ₂ -8-, -CH ₂ -CH ₂ -, -CH = CH- (cis and trans), -CHCH ₂ -, -CH (OH) CH ₂ - and -CH ₂ 8O-, by methods well known in this area. The directed substitution of one or more amino acids by a consensus sequence with an α-amino acid of the same type (for example, β-lysine instead of β-lysine) can be used in some embodiments to produce more stable peptides. In addition, spatially limited peptides containing a consensus sequence or a substantially identical variant of the consensus sequence can be obtained by methods known in the art (Mox & 01egax, 1992, App. Wow. Wuxet. 61: 387, incorporated herein by reference for any purpose); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges that cyclize this peptide.

As used herein, the terms antibody or antibody peptide (s) refer to a monomeric or multimeric protein containing one or more polypeptide chains. An antibody may specifically bind to an antigen and may have the ability to inhibit or modulate the biological activity of an antigen. Antibodies include natural antibodies, which are described below. In some embodiments, antibodies are prepared by recombinant DNA methods. In further embodiments, antibodies are prepared by enzymatic or chemical cleavage of natural antibodies. Antibodies include, but are not limited to, E (a b) -, E (a b ') -, E (a b') ₂ -, Εν - and single-chain Εν-fragments, as well as single-chain, chimeric, humanized, fully human, polyclonal and monoclonal antibodies. The antibody, which is understood here, as a minimum, includes a polypeptide that can specifically bind to the antigen, including all or part of the variable region of the light or heavy chain.

The variable region includes at least three complementarity determining regions of the heavy or light chains (CEB, also known as hypervariable regions, designated as CEB1, CEB2 and CEB3 according to KaBa1 e1 a1., 1991, 8ec. | Riys NeaIy 8g \ zse Ν.ΙΠ., VeShekia, ΜΌ; see also Cio1aa apy Beck, 1987, 1. Mo1. Vu1. 196: 901-17; Cio1aa1 a1., 1989, No. 1 Shge 342: 877-83) embedded in the frame section (the frame sections 1-4 are indicated: EB1, EB2, EB3 and EB4 according to KaLa1 e1 a1., above; see also CbO1aa apy Beck, above). The sections of the CMEA and frame segments are located as follows, starting from the amino terminus of the variable region: EB1-SEB1-EB2COV2-EV3-COV3-EB4.

The primary sequences of the framework regions of the variable regions of antibodies have a small amount of residues that are conserved in all types. However, many residues are highly conserved within types and / or within species and / or types, and many positions in antibodies are usually occupied by one of the known groups of amino acids. See KaLa1 e1 a1., Above. Or, the sequence can be recognized as an antibody by its predicted tertiary structure. The tertiary structure of the variable regions, which includes 9 β-chains forming a structure known as the β-barrel Ogeek keu, is extremely conservative, and the CMEA positions within this structure are also highly conservative. See, for example, Vogk e1 a1., 1994, 1. Mo1. Vu1. 242: 309-20; NipkarSheg apy Nooy, 1989, Ice. Ittyo1. 44: 163; ^ 1Shatk apy Vags1au, 1988, App. Weight. Ittyo1. 6: 381-405; Cio1aa apy Beck, above; KaLa1 e1 a1., Above.

The tertiary structure can be predicted using various computer programs, such as, for example, ОЕЕЕОБЭ® (Тпрок, Шс., 81. Бошк, МО; Сойх | кпй 8ко1шк, 1992, Proc. No. 11. Asai. -12102; Soikh | to e1 a1., 1992, 1. Mo1. Vu1. 227: 227-38; Soikh | to e1 a1., 1993, PrgoeS Eppd. 6: 801-10), a protein sorting program that overlays the analyzed the protein sequence for structural samples from Prgoel ea1a Vapk (REV) (Vegtap e1 a1., 2000, No1 Ac1yk Century 28: 235-242; 1agokhe \ skk | e1 a1., 1998, Prgo1. 8cg 7: 1431-1440) . In order to use OEIEEOBE® to classify a new amino acid sequence, this sequence is entered into a program that determines the probability score, which reflects how well it folds into previously known protein structures (matrix structures) that are present in the OEIEEOBE® database. For

- 14 011876 scoring ΟΕΝΕΕΟΕΌ® takes as a basis the similarity of the primary amino acid sequence, hidden samples of residues, local interactions and comparisons of the secondary structure. The ΟΕΝΕΕΟΕΌ® program superimposes the amino acid sequence on all matrix structures (or iterates over) in an existing database of protein folds, which includes the allowed structures for a number of antibodies. The ΟΕΝΕΕΘΕΌ® result is three lists of proteins from a database whose tertiary structures are most likely to be determined by the introduced amino acid sequence. Three lists contain three different scoring systems, calculated based on: (ί) only sequences, (,) sequences plus local conformation preferences plus hidden elements and (ίίί) sequences plus local conformation preferences plus hidden elements plus secondary structure. In each case, this program determines the optimal comparison, calculates the probability (P-value) that this degree of comparison was random, and represents the inverse of the P-value in the form of a scale with 999.9 (9.999x10 ² ), which is the highest possible score . Thus, the highest score indicates the least likelihood that the match was random. Thus, these scores reflect the degree to which the new protein coincides with different reference structures, and are useful in establishing whether a new protein belongs to members of a known protein family. For example, a sequence having an antibody variable region structure is expected to be comparable to at least one known variable region of an antibody with a sufficiently high P value, for example at least about 200, 300, 400, 500, 600, 700, 800, or higher.

The term heavy chain includes any immunoglobulin polypeptide having a sufficient sequence of the variable region to ensure the specificity of binding of γ-γ. The term light chain includes any immunoglobulin polypeptide having a sufficient sequence of the variable region to ensure the specificity of binding of γ-γ. Such a heavy or light chain may, but need not, bind to ΙΕΝ-γ in the absence of a light chain if it is a heavy chain, or a heavy chain if it is a light chain. The full-sized heavy chain includes the variable region domain: ν _Η , and three constant region domains: C _n 1, Cc2 and C _n 3. The νιι domain is at the amino terminus of the polypeptide, and the C _n 3 domain is at the carboxy terminus. As used herein, the term heavy chain encompasses a full-sized heavy chain and fragments thereof. The full-sized light chain includes the domain of the variable region: and the domain of the constant region: Cb. Like the heavy chain, the variable region domain of the light chain is at the amino terminus of the polypeptide. As used herein, the term light chain encompasses a full-sized light chain and fragments thereof. The E (ab) fragment consists of one light chain and C _n 1 and the variable regions of one heavy chain. The heavy chain of an E (a b) molecule cannot form a disulfide bond with a molecule of another heavy chain. E (ab ') - the fragment contains one light chain and one heavy chain, which contains more constant regions between the Cn1 and Cn2 domains, so that an interchain disulfide bond can form between two heavy chains with the formation of an E (ab') ₂ molecule . The Εν site includes the variable regions of both the heavy and light chains, but does not have constant regions. Single chain antibodies are Εν molecules in which the variable regions of the heavy and light chains are connected by a flexible linker to form a single polypeptide chain that forms the antigen-binding site. Single chain antibodies are discussed in detail in the publication of the international patent application νθ 88/01649 and patents I8 4946778 and 5260203.

The invention also encompasses fully human, humanized and chimeric antibodies. As implied herein, fully human antibodies include amino acid sequences encoded only by polynucleotides, which ultimately are of human origin, or amino acid sequences that are identical to such sequences. As implied here, antibodies encoded by the human immunoglobulin-coding DNA inserted into the murine genome produced in the transgenic mouse are fully human antibodies, since they are encoded by DNA, which ultimately has human origin. In this situation, the human immunoglobulin-coding DNA can be rearranged (to encode the antibody) within the mouse and somatic mutations can also occur. Antibodies, originally encoded by human DNA, which undergoes such changes in the mouse, are fully human antibodies, as implied here. The use of such transgenic mice allows the selection of fully human antibodies relative to the human antigen. In nature, in most cases this is impossible, since the human immune response against their own antigen is not normally observed. One skilled in the art will understand that fully human antibodies are useful as therapeutic agents, especially in the treatment of chronic diseases, as they are unlikely to elicit an immune response against themselves. In contrast, many non-human antibodies are known to enhance the immune response against themselves when used in humans; a situation that makes the continuous use of such antibodies in humans impractical. Thus, fully human antibodies solve the long-standing problem that is encountered when using antibodies to treat chro

- 15 011,876 conditions, including human diseases. See, for example, B1Shai, 1988, 1ttypo1. Tobau 9: 3740; Note £$ е1 а1., 1991, С1ш. 1ttypo1. & 1tiporaLo1. 59: 89-103; T) apyga e1 a1., 1990, 1tipo1 & Ce11 B1o1. 68: 367-76. Thus, fully human anti-γ antibodies are particularly well suited for the treatment of chronic human diseases associated with γ-γ, such as autoimmune diseases.

In a humanized antibody, the complete antibody, with the exception of the SEY sites, is encoded by a polynucleotide of human origin or is identical to such an antibody, except for its SAY. CEIs, which are encoded by nucleic acids that are not from the human body, are transplanted into the framework in the form of a β-layer of the variable region of a human antibody to create an antibody whose specificity is determined by the transplanted CEI. The creation of such antibodies is described, for example, in XVO 92/11018, 1ope8 e1 a1., 1986, No. 11ige 321: 522-25, Ugoeuep e1 a1., 1988, 8c1epse 239: 1534-36. This work emphasizes the key importance of SEC plots in the formation of an antigen-binding site. A chimeric antibody contains a constant region of a person (which is encoded by a polynucleotide of human origin or is identical to such a constant region of a person) and a non-variable region, which is human. The creation of such antibodies is described, for example, in patent I8 5681722.

It is understood that a divalent antibody other than a multispecific or multifunctional antibody, in some embodiments, contains binding sites having identical antigenic specificity.

Evaluation of antibody binding and specificity according to the invention: the antibody specifically binds and / or essentially inhibits the adhesion of ΙΡΝ-γ at its receptor when an excess of antibody reduces the number of receptors associated with ΙΡΝ-γ, or, conversely, by at least about 20 , 40, 60, 80, 85% or more (according to the results of measurements in the analysis of competitive binding w νίΙΐΌ). It can be expected that a specifically bound antibody will have an equilibrium dissociation constant of binding to ΙΡΝ-γ, which is less than or equal to 10 ⁻⁸ mol, possibly less than or equal to 10 ⁻⁹ or 10 ⁻¹⁰ mol.

For therapeutic use, an important characteristic of an anti-ΙΡΝ-γ antibody is whether it can inhibit or modulate the biological activity of ΙΡΝ-γ. ΙΡΝ-γ has many different biological effects that can be determined in many different assays in different types of cells. The ability of an anti-ΙΡΝ-γ antibody to inhibit or modulate the biological activity of ΙΡΝ-γ can be determined using the A549 assay described in Example 6 below, or using a similar assay that determines the ability of an antibody to remove the inhibition of cell proliferation observed in the presence of ΙΡΝ- γ. For this assay to produce meaningful results, the proliferation of cells used in this assay must be inhibited by the ΙΡΝ-γ used in this assay. Human ΙΡΝ-γ can inhibit the proliferation of certain types of cells, including A549 cells (examples 6 and 7). Mouse ΙΡΝ-γ can inhibit the proliferation of ΚΑ \ ν 264.7 cells (Example 7), but not of A549 cells. In particular, when testing the ability of an antibody to inhibit or modulate the biological activity of non-human ΙΡΝ-γ, cell types other than A549 cells can be used, since non-human ΙΡΝ-γ may be able to inhibit the proliferation of A549 cells or may not have that ability. Not every antibody that specifically binds to an antigen can block the binding of an antigen to its normal receptor and thus inhibit or modulate the biological effects of an antigen when bound to its receptor. As is known in the art, this effect may depend on which portion of the antigen the antibody binds to, as well as the absolute and relative concentrations of antigen and antibody, in this case ΙΡΝ-γ and anti-ΙΡΝ-γ antibody. In order for an antibody to be considered as capable of inhibiting or modulating the biological activity of ΙΡΝ-γ, as implied here, it must be able to remove the inhibition of cell proliferation observed in the presence of ΙΡΝ-γ, as determined using fluorescence in analysis A549 (example 6) or a similar analysis, at least about 20, 40, 60, 80, 85, 100% or more, when the concentration of ΙΡΝ-γ is in the range, for example, about about EU ₈₀ or EU ₉₀ , where the effects of an agent that inhibit its biological activity can be easily detected. The EU ₈₀ , as understood here, is the amount of ΙΡΝ-γ necessary for 80% of the maximum ΙΡΝ-γ effect to be observed. If the concentration of ΙΡΝ-γ is significantly higher than EC90, then the effects of the inhibitory agent may be less obvious due to the excess of ΙΡΝ-γ. The antibody concentration required to inhibit or modulate the biological activity of ΙΡΝ-γ can vary greatly and may depend on how strongly this antibody binds to ΙΡΝ-γ. For example, to inhibit or modulate biological activity in an A549 assay, one antibody molecule or less than one per ΙΡΝ-γ molecule may be sufficient. In some embodiments, a ΙΡΝ-γ: antibody ratio of about 2: 1, 1: 1, 1: 2, 1: 4, 1: 6, 1: 8, 1:10, 1:20, 1:40 may be necessary. , 1:60, 1: 100 or 1: 50,000, to inhibit or modulate the biological activity of ΙΡΝ-γ, if the concentration of ΙΡΝ-γ is from about EC50 to at

- 16 011876 measured by the EU ₉₀ . Relations between IΕN-γ antibodies between these values are also possible.

In further embodiments, antibody variants may include antibodies comprising a modified Ec fragment or a modified heavy chain constant region. The ec fragment, which means the fragment that crystallizes, '' or the constant region of the heavy chain can be modified by a mutation in order to give the antibody other characteristics (see, for example, Bebop apb / UooG. 1992, Abhapsek ίη Lptipokod 51: 1-84; Eauslcn apb Vo11apb, 2001, Appi. Keu. Itipo1. 19: 275-90; 8e1bk e! A1., 2001, 1o1 ; Mebekap e! A1., 1998, Eig. I. Ittiηο1. 28: 2092-2100; which are all hereby incorporated by reference). Such mutations may include substitutions, insertions, deletions, or any combination thereof and are usually carried out by site-directed mutagenesis using one or more mutagenic oligonucleotides by the methods described here, as well as by methods known in the art (see, for example, Mashabk e! A1 ., M0Eesiak CE0MX6: EAB0KAT0KU MAHEak, 3GB Eb., 2001, Co1b 8rgd Nagb, KU. And Vegdeg apb K1tte1, METN0B8 W ENΖΥМΟ ^ ΟCb, Wlite 152, Sbkеbе1e1. ^ o, CA., which are incorporated herein by reference).

In some embodiments, antibody variants include glycosylated variants, wherein the amount and / or type of glycosylation site is altered compared to the amino acid sequences of the parent polypeptide. In some embodiments, protein variants include more or less ^ linked glycosylation sites than the native protein. The linked glycosylation site is characterized by the sequence Akp-X-8eg or Akp-X-Tb, where the amino acid residue denoted by X can be any amino acid residue, except proline. Substitution of amino acid residues with the formation of this sequence provides a new site that is potential for adding ^ linked carbohydrate chain. Or, substitutions that exclude this sequence will remove the already existing ^ linked carbohydrate chain. A rearrangement of ^ linked carbohydrate chains is also proposed, where one or more ^ linked glycosylation sites (usually those that are natural) are eliminated and one or more new ^ linked sites are created. Additional preferred antibody variants include cysteine variants, where one or more cysteine residues are deleted or replaced by another amino acid (e.g., serine) compared to the parent amino acid sequence. Cysteine variants may be useful when antibodies need to be folded back into a biologically active conformation, for example, after isolation of insoluble Taurus inclusions. Cysteine variants usually have fewer cysteine residues than the native protein, and typically have an even amount to minimize interactions of unpaired cysteines.

The term agent is used herein to mean a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.

As used herein, the terms labeled or labeled refer to the incorporation of a detectable marker, for example, by incorporating an isotope-labeled amino acid or attaching to a polypeptide or nucleic acid a fluorescent marker, a chemiluminescent marker or an enzyme having detectable activity, or attaching biotin moieties to the polypeptide that can be detected by the labeled avidin (e.g., streptavidin, preferably comprising a detectable marker, such as a fluorescent marker, chemiluminescent marker, or enzymatic activity, which can be detected, in particular, by optical or colorimetric methods). In some embodiments, the label may also be therapeutic. Various methods for labeling polypeptides and glycoproteins are known in the art and can advantageously be used in the methods disclosed herein. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g. ³ H, ¹⁴ C, ¹⁵ K ³⁵ δ, ⁹⁰ Υ, ^{99 t} Tc, ^w Tp, ¹²⁵ Ι, ¹³¹ Ι), fluorescent labels (e.g. , fluorescein isothiocyanate or FITC, rhodamine or lanthanide phosphors), enzyme labels (e.g. horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, hapten labels, such as biotinyl repeatable groups, and e.g. paired series atelnosti leucine zipper (1eiste / 1rreg), binding sites for secondary antibodies, metallosvyazyvayuschie domains, or epitope tags (! ADK)). In some embodiments, the labels are connected by spacer legs (such as (CH ₂ ) _n , where n is less than about 20) of different lengths to reduce possible steric mismatch.

The term biological sample, as used herein, includes, but is not limited to, any amount of a substance from a living organism or an organism that has ceased to live. Such living organisms include, but are not limited to, humans, mice, monkeys, rats, rabbits, and other animals. Such substances include, but are not limited to, blood, serum, urine, cells, organs, tissues, bone, bone marrow, lymph nodes, and skin.

As used herein, the term “pharmaceutical agent or drug” refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly

- 17 011876 administration to the patient.

The term “IΡN-γ mediated disease” includes, but is not limited to, inflammatory, infectious, and autoimmune diseases. As used herein, the term autoimmune disease refers to painful conditions and conditions where the patient’s immune response is directed against the patient’s own elements. For example, IΡN-γ mediated diseases include, but are not limited to, Acquired Immunodeficiency Syndrome (AIDS), rheumatoid arthritis, including juvenile rheumatoid arthritis; inflammatory bowel disease, including ulcerative colitis and Crohn's disease; multiple sclerosis, Addison’s disease, diabetes (Ι type), epididymitis, glomerulonephritis, Graves disease, Guillain-Barré syndrome, chronic lymphomatous thyroiditis, hemolytic anemia, systemic lupus erythematosus (SLE), lupus nephritis, asthenic bulbar palsy psoriasis, p arthritis, atherosclerosis, erythropoietin resistance, transplant versus host, transplant rejection, autoimmune hepatitis-induced liver damage, biliary cirrhosis, alcohol-induced liver damage, including alcoholic cirrhosis, rheumatism, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, including ankylosing spondylitis, thyroiditis and vasculitis. The term disease mediated by GR # gamma also encompasses any medical condition associated with increased levels of GE # or increased sensitivity to GE #.

Treating an IΡN-γ mediated disease, including autoimmune disease, involves alleviating at least one symptom of the disorder, reducing the severity of the disease, or slowing down or preventing the development of a more serious disease that occurs with some frequency after treatment of the condition. Treatment should not imply that the disease is completely cured. A useful therapeutic agent is only required to reduce the severity of the disease, reduce the severity of the symptom or symptoms associated with the disease or its treatment, or to provide an improvement in the quality of life of the patient, or to slow down the development of a more serious disease that can occur with some frequency after treatment of the condition. For example, if the disease is rheumatoid arthritis, the therapeutic agent can reduce joint swelling, reduce the number of affected joints, or slow down or inhibit bone loss. A patient with SLE may have symptoms along with other symptoms such as skin lesions, fever, weakness, arthritis, swollen lymph nodes, pleurisy, pericarditis, and / or anemia. Such symptoms can be evaluated by any of a number of standard methods, including, for example, visual observation, photographing, measuring temperature, grip strength or joint size and / or microscopic examination of the blood to determine the concentration of red blood cells. The present invention encompasses a method of treatment, comprising administering to a patient suffering from an IΡN-γ mediated disease, anti-GEnu antibodies of the invention in an amount and for a time sufficient to induce sustained improvement over an initial level of indicator that reflects the severity of a particular disorder or the severity of the symptoms caused by this disorder, or to slow down or prevent the development of a more serious disease that follows after treating the condition in some or all cases x The invention does not exclude the possible treatment with other therapeutic agents before, after and / or during treatment with an antibody to ΙΤ ^ γ.

As used herein, the term “substantially pure or substantially purified” means a compound or variety that is the predominant variety (i.e., on a molar basis, it is more widespread than any other individual variety in the composition). In some embodiments, the substantially purified fraction is a composition wherein the variety comprises at least about 50% (on a molar basis) of all macromolecular species present. In some embodiments, a substantially pure composition will contain more than about 80, 85, 90, 95, or 99% of all macromolar species present in the composition. In some embodiments, this species is purified to substantial homogeneity (contaminant species cannot be determined in the composition by standard detection methods), where the composition consists essentially of one macromolecular species.

The term patient refers to humans and animals.

Unless otherwise indicated, singular terms also encompass the plural.

Since GE # is a cytokine with many functions, including protecting the body from a viral infection and regulating some aspects of the immune response, the increased activity of HEDγ can contribute to some pathological conditions. According to some embodiments of the invention, antibodies directed against IΡN-γ can be used to treat diseases mediated by IΡN-γ, including but not limited to the diseases mentioned above.

In one aspect of the invention, fully human anti-human monoclonal antibodies are provided and have biological and immunological specificity for binding to human IΡN-γ. Variable regions (8EC ΙΌ N0: 6, 8EC ΙΌ N0: 8, 8EC ΙΌ N0: 10, 8ES GO N0: 12, 8ES GO N0: 14, 8EP GO N0: 16, 8ES GO N0: 30 and 8ES GO N0: 31 ) included in such

- 18 011876 antibodies, full heavy and light chains of such antibodies (8EC ΙΌ ΝΟ: 17, 8EC ΙΌ ΝΟ: 18, 8EC ΙΌ ΝΟ: 19, 8EC ΙΌ ΝΟ: 20, 8EC ΙΌ ΝΟ: 21 and 8EC ΙΌ ΝΟ: 22) and antibodies comprising specific sites of SOW (SEEV SEV2 and / or SEV3 heavy and light chains; 8EC ΙΌ ΝΟ: 34-8EC ΙΌ ΝΟ: 44) are included in the scope of this invention. Specific embodiments of this aspect of the invention are sequences corresponding to sections of SOW, especially sections of SEV1-SEV3, heavy and light chains proposed in the invention. In addition, the invention encompasses antibodies containing CMEA3-sequence disclosed here (8EC ΙΌ ΝΟ: 36, 8EC ΙΌ ΝΟ: 37, 8EC ΙΌ ΝΟ: 43 and / or 8EC ΙΌ ΝΟ: 44), which may also contain sequences of at least at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identical to any of the variable region sequences or complete heavy or light chain sequences disclosed herein, where this antibody can inhibit or modulate the biological activity of ΙΡΝ-γ.

In another aspect of the invention, isolated nucleic acids or polynucleotides encoding the antibodies of the invention are provided. Antibodies of the invention can specifically bind to γ-γ and / or inhibit or modulate the biological activity of γ-γ. In particular, the invention encompasses polynucleotides comprising nucleotide sequences encoding the amino acid sequences 5EO ΙΌ ΝΟ: 17, 5EO ΙΌ ΝΟ: 18, 5EO ΙΌ ΝΟ: 19, 5EO ΙΌ ΝΟ: 20, 5EO ΙΌ ΝΟ: 21, 5EO ΙΌ ΝΟ: 22, 5EO ΙΌ ΝΟ: 32, 5EO ΙΌ ΝΟ: 33, 5EO ΙΌ ΝΟ: 5, 5EO ΙΌ ΝΟ: 7, 5EO ΙΌ ΝΟ: 9, 5EO ΙΌ ΝΟ: 11, 8EC ΙΌ ΝΟ: 13, 8EC ΙΌ ΝΟ: 15, 8EC ΙΌ ΝΟ: 30, 8EC ΙΌ ΝΟ: 31 and / or sequences that are at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identical to these sequences, where comparison of the above sequences and the test sequence and covers at least about 50, 60, 70, 80, 90 or 100 amino acids. In addition, the invention provides polynucleotides containing 8EC ΙΌ ΝΟ: 45, 8EC ΙΌ ΝΟ: 46, 8EC ΙΌ ΝΟ: 47 and / or 8EC ΙΌ ΝΟ: 48, encoding antibodies that can specifically bind to ΙΡΝ-γ and / or inhibit or modulate the biological activity of ΙΡΝ-γ. In addition, the invention encompasses polynucleotides that are at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% identical to 5EO ΙΌ ΝΟ: 5, 5EO ΙΌ ΝΟ: 7 , 5ЕО ΙΌ ΝΟ: 9, 5ЕО ΙΌ ΝΟ: 11, 5ЕО ΙΌ ΝΟ: 13, 5ЕО ΙΌ ΝΟ: 15, 5ЕО ΙΌ ΝΟ: 30, 5ЕО ΙΌ ΝΟ: 31, 5ЕО ΙΌ ΝΟ: 17, 5ЕО ΙΌ ΝΟ: 18, 5ЕО ΙΌ ΝΟ: 19, 5EO ΙΌ ΝΟ: 20, 5EO ΙΌ ΝΟ: 21, 8EC ΙΌ ΝΟ: 22, 8EC ΙΌ ΝΟ: 32 or 8EC ΙΌ ΝΟ: 33, moreover, an antibody encoded partially by each of these polynucleotides can inhibit or modulate the biological activity and / or specifically bind to ΙΡΝ-γ, and where the nucleotide sequence matching, As indicated immediately above, and the test sequence covers at least about 100, 150, or 200 nucleotides.

In the table. 2 provides a brief description of the sequences and their relationship to the sequence identification numbers.

table 2

Summary of Sequence Listing

Sequence Identification Number Short description 5Е0 GO N0: 1 The nucleotide sequence encoding the constant region of the heavy chain of the antibody 1118, 1118 *, 1119, 1121 or 1121 * 5Е0 GO N0: 2 Amino acid sequence of the constant region of the heavy chain of the antibody 1118, 1118 *, 1119, 1121 or 1121 * ZEO GO N0: 3 The nucleotide sequence encoding the constant region of the light chain of the antibody 1118, 1118 *, 1119, 1121 or 1121 * ZEO> 0 N0: 4 Amino acid sequence of the constant region of the light chain of the antibody 1118, 1118 *, 1119, 1121 or 1121 * ZEO GO N0: 5 The nucleotide sequence encoding antibody 1119 heavy chain variable region ZEO GO N0: 6 Amino acid sequence of the variable region of the heavy chain of the antibody 1119 ZEO GO N0: 7 The nucleotide sequence encoding antibody 1119 light chain variable region ZEO GO N0: 8 Amino acid sequence of the variable region of the light chain of antibody 1119 ZEO GO N0: 9 The nucleotide sequence encoding antibody 1118 heavy chain variable region ZEO GO N0: 10 Amino acid sequence of the variable region of the heavy chain of the antibody 1118 ZEO GO N0: 11 The nucleotide sequence encoding antibody light chain variable region 1118 or 1118 * ZEO GO N0: 12 Amino acid sequence of the variable region of the light chain of the antibody 1118 or 1118 * ZEO GO N0: 13 The nucleotide sequence encoding antibody heavy chain variable region 1121 or

- 19 011876

1121 *

5VD GO N0: 14 Amino acid sequence of the variable region of the heavy chain of the antibody 1121 or 1121 * 8Εζ) GO N0: 15 The nucleotide sequence encoding antibody 1121 light chain variable region ZER ΙΡ N0: 16 Amino acid sequence of the variable region of the light chain of antibody 1121 8E0 GO N0: 17 Amino acid sequence of the complete heavy chain of antibody 1119 8E () GO N0: 18 Amino acid sequence of the complete light chain of antibody 1119 8E0 GO N0: 19 Amino acid sequence of the complete heavy chain of antibody 1118 8Е0 GO N0: 20 Amino acid sequence of the complete light chain of antibody 1118 or 1118 * 8E <3 GO N0: 21 Amino acid sequence of the full heavy chain of antibodies 1121 or 1121 * 8E0 GO N0: 22 Amino acid sequence of the complete light chain of antibody 1121 8E0 GO N0: 23 The nucleotide sequence of the PCR primer YOEO GO N0: 24 The nucleotide sequence of the PCR primer 8EC GO N0: 25 The nucleotide sequence of the PCR primer 8EC GO N0: 26 The nucleotide sequence of the PCR primer 8E0 GO N0: 27 The nucleotide sequence of the PCR primer 5Е0 GO N0: 28 The nucleotide sequence of the PCR primer P ν / L TTL \ T / L-L <* \ nucleotide "sequence of the NCG primer 8E <E GO N0: 30 Amino acid sequence of the variable region of the heavy chain of the antibody 1118 * 8Εζ> GO N0: 31 Amino acid sequence of the variable region of the light chain of the antibody 1121 * 5E (,> GO N0: 32 Amino acid sequence of the complete heavy chain of the antibody 1118 * 8E <2 U N0: 33 Amino acid sequence of the complete light chain of antibody 1121 * 8E <) GO N0: 34 The amino acid sequence of the C0K1 heavy chain of antibody 1119, 1118, 1118 *, 1121 or 1121 * ZEO GO N0: 35 The amino acid sequence of SCK2 of the heavy chain of antibody 1119, 1118, 1118 *, 1121 or 1121 * 8ES) GO N0: 36 Amino acid sequence of SVKZ heavy chain antibodies 1119 8E <2 GO N0: 37 Amino acid sequence of JUICE.Z heavy chain antibodies 1118,1118 *, 1121 or 1121 * 5ES2 GO N0: 38 Amino acid sequence of C0P, 1 light chain antibodies 1119 or 1121 5EO GO N0: 39 Amino acid sequence of SOYA light chain antibodies 1118 or 1118 * 8E <) GO N0: 40 Amino acid sequence of SOK1 light chain antibodies 1121 * 5ΕςΐϋΝ0: 41 Amino acid sequence of SEC2 light chain antibodies 1119, 1118,1118 * or 1121 8E0 GO N0: 42 Amino acid sequence of SOK2 light chain antibodies 1121 * 8E0 GO N0: 43 Amino acid sequence of SOKZ light chain antibodies 1119, 1118, 1118 * or 1121 8EC GO N0: 44 Amino acid sequence of SOKZ light chain antibodies 1121 * 8E0 GO N0: 45 The nucleotide sequence encoding the SOKZ heavy chain antibodies 1P 9 8EC GO GO N0: 46 The nucleotide sequence encoding the SOKZ heavy chain antibodies 1118, 1118 *, 1121 or 1121 * Yaer GO N0: 47 The nucleotide sequence encoding the SOKZ light chain antibodies 1118, 1118 *, 1119 or 1121 8E <2 GO N0: 48 Amino acid sequence immediately preceding the heavy chain C0K1 8E0 GO N0: 49 Amino acid sequence that can directly precede the C0K2 heavy chain 8EC GO 0: 50 Amino acid sequence that almost always follows the heavy chain SOKZ 8E0 GO N0: 51 Amino acid sequence that usually follows the light chain SOKZ 8E0 10 N0: 52 Amino Acid Signal Sequence sequences 5Е0 GO N0: 53 Amino Acid Signal Sequence sequences 8E () GO N0: 54 Amino Acid Signal Sequence sequences 8E0 GO N0: 55 Amino Acid Signal Sequence sequences 8E0 GO N0: 56 The nucleotide sequence of the variable region of the heavy chain of the antibody 1118 * 8E0 GO N0: 57 The nucleotide sequence of the variable region

(light chain antibodies 1121 *

- 20 011876

In yet another aspect of the invention, hybridoma cells and cell lines that express immunoglobulin molecules and antibodies of the invention, possibly monoclonal antibodies, are provided. In an additional aspect, a hybridoma cell or a cell from a cell line that expresses and / or secretes an immunoglobulin molecule or an antibody of the invention can be implanted into a patient, whereby an antibody of the invention or an immunologically functional immunoglobulin fragment thereof is expressed and / or secreted in a patient, inhibiting or thus modulating the activity of ΙΕΝ-γ.

The invention also provides biologically and immunologically purified preparations of antibodies, preferably monoclonal antibodies, against β-γ, and having biological and immunological specificity with respect to specific binding to human β-γ.

The ability to clone and reproduce very large human loci in artificial yeast chromosomes (ULS) and introduce them into the mouse germ line allows us to develop a useful approach to elucidating the functional components of very large or roughly mapped loci, as well as to obtain useful models of human diseases. Moreover, the use of such technology to replace mouse loci with their human equivalents provides a unique opportunity to gain insight into the expression and regulation of human gene products during development, their relationship with other systems and their involvement in the induction and development of the disease.

An important practical application of such a strategy is to change the humoral immune system of a mouse by introducing human immunoglobulin (1e) loci to mice in which endogenous 1d genes have been inactivated (international application νθ 93/12227). This system makes it possible to study the mechanisms underlying the programmed expression and assembly of antibodies, as well as their role in the development of B cells. Moreover, such a strategy offers a source of production of fully human monoclonal antibodies (mAbs). Fully human mAbs are expected to minimize the immunogenic and allergic responses inherent in murine mAbs or mAbs derived from murine, and thereby increase the efficacy and safety of the administered antibodies. Fully human antibodies can be used in the treatment of chronic and recurrent human diseases, such as osteoarthritis, rheumatoid arthritis and other inflammatory conditions, the treatment of which requires repeated administration of antibodies. Thus, one particular advantage of the anti-ΙΕΝ-γ antibodies of the invention is that these antibodies are fully human and can be administered to patients without stress to the body, although the use of murine anti-human antibodies, or other non-fully human antibodies previously described, or non-human antibodies from non-human species, is usually associated with the need to minimize adverse reactions.

Using the methods described herein, one of skill in the art can construct murine strains defective in the production of murine antibodies with large fragments of human 1d loci so that such mice produce human antibodies and not produce murine antibodies. Large human S-fragments can support a wide variety of variable genes, as well as the proper regulation of antibody production and expression. Using the mouse cell apparatus to ensure diversity and selection of antibodies and the lack of immunological tolerance to human proteins, it is possible to obtain a repertoire of human antibodies in these mouse strains that provides high affinity antibodies against any antigen of interest, including human antigens. Using hybridoma technology, antigen-specific human mAbs with the desired specificity can be obtained and selected.

In some embodiments, a person skilled in the art may use constant regions from species other than humans, together with the human variable region (s) in such mice, to produce chimeric antibodies.

The natural structure of antibodies

Most natural antibody structural units are usually tetramer. Each such tetramer usually consists of two identical pairs of polypeptide chains, each pair having one full-sized light chain (usually having a molecular weight of about 25 kDa) and one full-sized heavy chain (usually having a molecular weight of about 50-70 kDa). The amino terminal portion of each light and heavy chain typically includes a variable region of about 100-110 or more amino acids, which is usually responsible for antigen recognition. The carboxy-terminal portion of each chain usually defines a constant region responsible for effector function. Human light chains are usually divided into kappa and lambda light chains. Heavy chains are usually classified as mu, delta, gamma, alpha or epsilon and the antibody isotype is defined as 1dM, Ι§Ι, 1§C. 1dL and Ι§Ε, respectively. ΙβΟ has several subclasses, including, but not limited to, 01§01, Ι§02, ΙβΟ3 and ΙβΟ4. Ι§Μ includes subclasses, but not limited to, [dM 1 and Ι§Μ2. Ι§Λ is likewise subclassed, including, but not limited to, Ι§Ά 1 and Ι§Ά2. Within the full-sized light and heavy chains, the variable and constant regions are usually connected by a region of about 12 or more amino acids, where the heavy chain also contains a Ό '' region of

- 21 011876 measure 10 or more amino acids. See, for example, ΕϋΝΟΆΜΕΝΤΑΕ ΙΜΜυΝΟΕΟΟΥ, SI. 7, 2 ' ¹ eb. (Rai1, V., eb.), 1989, Vausp Rgekk, Ν.Υ. (incorporated by reference in its entirety for all purposes). The variable regions of each light / heavy chain pair typically form an antigen binding site.

Some natural antibodies found in camels and llamas are dimers consisting of two heavy chains and free of light chains. Μυ ^ ι ^ αηκ e! A1., 2001, 1. Vue! Expo1. 74: 277-302; OektuZeg e! A1., 2001, 1. Βίοί. SIET. 276: 26285-90. The invention encompasses dimeric antibodies consisting of two heavy chains that can bind to β-γ and / or inhibit the biological activity of β-γ. A crystallographic study of a camelid antibody found that heavy-chain CMEA3, having 19 amino acids in length, forms a surface that interacts with the antigen and covers two other hypervariable regions (EectuSeg e! A1., Above). Thus, CEB3 is important for antigen binding in dimeric camel antibodies, as well as in more typical tetrameric antibodies.

Variable regions usually have the same general structure where conservative framework regions (EBs) are connected by three hypervariable regions, also called complementarity determining regions, or CMEAs. SIV regions from two chains of each pair are usually immersed inside the framework regions, which allows binding to a specific epitope. The variable regions of both light and heavy chains usually contain, from the конца-end to the C-end, domains EB1, CEB1, EB2, CEB2, EB3, CEB3 and EB4. The amino acids in each domain are usually distributed according to the interpretation given in KaLa! et al., which is explained in more detail below (Kaaa! e! a1., 8c. | unsex oG Proof! oo 1xpx1o1a1e1eq ((1991, No. 1bopa1 1pxucoe oG NeaIb, Veshekba, Μ6.); see also & Lekk, 1987, 1. Mar. Vu1. 196: 901-917; C1i11ia e! A1., 1989, No. III1 342: 878-883). The CMEA sites create the main surface contact points for antigen binding. See, for example , Clu11ia apb leck, above. In addition, light chain CE3 and especially heavy chain CE3 can form the most important determinants when antigen is bound within the variable regions of the light and heavy chains. See, for example, C1u11ia apb lek. k, above; PeDbegu e! a1. (2001), 1. Μοί. Vu1. 310: 603-15; Chi apb Jau1k (2000), Ιιηιηιιιάν 13 (1): 37-45; EektuZeg e! a1. (2001) , 1. Vu1. C1et. 276 (28): 26285-90; and Μ ί ί та ^ η η η κ (2001), 1. Vu! Expo 1. 74 (4): 277-302. In some heavy chain SEV3 antibodies, apparently forms the main area of contact between the antigen and the antibody (EektuZeg e! a1., above). Selection schemes t ίΙΐΌ v. in which only SEV3 changes, can be used to vary the binding properties of the antibody (Μ ί ^ ^ ^ η η выше, above; PeDbegu, above).

CMEA sites can be located in the sequence of variable regions of the heavy chain as follows. CMEA1 begins at approximately 31 mature antibodies and is usually about 5-7 amino acids in length, and almost always preceded by Suk-Xxx-Xxx-Xxx-Xxx-Xxx-Xxxxxxx-Xxx (8ΕΟ ΙΌ ΝΟ: 48) (where Xxx represents any amino acid). The residue after the CEB1 heavy chain is almost always tryptophan, often Tgr-Ya1, Tgr-11e or Tgr-A1a. Between the last residue in CMEA1 and the first in CMEA2 there are almost always 14 amino acids, and CMEA2 usually contains from 16 to 19 amino acids. CEB2 can immediately precede the sequence Ley-S1i-Tgr-11e-C1u (8ΕΟ ΕΟ ΕΟ: 49), and it can be directly followed by Luk / Agdiei / 11e / Va1 / Pb / Tb / A1a-Tb / §eg / 11e / A1a . Other amino acids may precede or follow SEV2. Between the last residue in CMEA2 and the first in CMEA3 there are almost always 32 amino acids, and CMEA3 may consist of about 3-25 residues in length. Suk-Xxx-Xxx almost always immediately precedes SIV3, and Tgr-C1u-Xxx-S1u (8ΕΟ ΙΌ ΝΟ: 50) almost always follows SIV3.

The CMEA portions of the light chain can be arranged in the sequence of the light chain as follows. SEB1 begins at about 24 residues of a mature antibody and usually has about 10-17 residues in length. It is almost always preceded by a souk. Between the last residue of CMEA1 and the first residue of CMEA2, 15 amino acids are almost always located, and CMEA2 almost always has 7 residues in length. SIV2 is usually preceded by Pe-Tug, Va1-Tug, Pe-Luk or Pe-Pb. Between SIV2 of the light chain and SIV3 there are almost always 32 residues, and SEV3 is usually about 7-10 amino acids in length. CMEA3 is almost always preceded by Suk, and it is usually followed by Pke-C1u-Xxx-C1u (8ΕΟ ΙΌ ΝΟ: 51).

One skilled in the art will recognize that the lengths of the framework regions surrounding the SIW may include inserts or deletions that make their length different from the typical length. As implied here, the length of the frame sections of the heavy chain is in the following ranges: EB1 amino acids 0-41; EB2 - amino acids 5-24; EB3 - amino acids 13-42 and EB4 - amino acids 0-21. In addition, according to the invention, it is assumed that the lengths of the frame sections of the light chain are in the following ranges: EB1 - 6-35 amino acids; EB2 - 4-25 amino acids; EB3 - 2-42 amino acids and EB4 - 0-23 amino acids.

Natural antibodies typically contain a signal sequence that directs the antibody to the cell pathway of protein secretion and which is not present in a mature antibody. A polynucleotide encoding an antibody of the invention can encode a native signal sequence or a heterologous signal sequence, as described below.

- 22 011876

Maturation of antibodies ίη νίΐτο

Antibodies can undergo maturation ίη νίΐτο, which leads to the formation of antibodies with altered properties, such as higher affinity for the antigen or lower dissociation constant. Change only residues within SEC, especially SEC3. can produce altered antibodies that bind to the same antigen, but with greater affinity. See, for example, 8cSet e1 a1., 1996, 1. Μοί. ΒίοΙ. 262: 551-67; Wapd e1 a1., 1995, 1. Μοί. ΒίοΙ. 254: 392-403. The invention encompasses antibodies created by a variety of ίη νίΐτο selection schemes, such as affinity maturation and / or shuffling (8Yi1td) chains (Kapd e1 a1., 1991, Proc. No. 11. Asab. 8sg 88: 11120-23), or shuffling DNA (81stet, 1994, No. 1ite 370: 389-391), in accordance with which antibodies having useful properties can be selected. In many schemes, a known antibody randomizes ίη νί1το to specific positions, often within an SEC, and undergoes a selection process, whereby antibodies with desired properties, such as increased affinity for a particular antigen, can be isolated. See, for example, νаη άеη Weiskem e1 a1., 2001, 1. Μοί. ΒίοΙ. 310: 591-601: E51bayu e1 a1., 2001, 1. Μοί. ΒίοΙ. 310: 603-15; ¥ apu e1 a1., 1995, 1. Μοί. ΒίοΙ. 254: 392-403; 8sets e1 a1., 1996, 1. Μοί. ΒίοΙ. 263: 55167. Typically, such mutant antibodies may contain several altered residues in one or more RNS, depending on the mutagenesis scheme and selection steps. See, for example, νаη άеη Βеiskеη e1 a1., Above.

Bispecific or bifunctional antibodies

A bispecific or bifunctional antibody is usually an artificial hybrid antibody having two different heavy chain / light chain pairs and two different binding sites. Bespecifically antibodies can be obtained in various ways, including, but not limited to, fusion of hybridomas or crosslinking of P (a b ') fragments. See, for example, δοηβδίνίΐηί & ^ asytηη, 1990, C1t. Exp ^ timk 79: 315-321; 81ο81ο1ηγ e1 a1., 1992, 1. Ittiηο1. 148: 1547-1553.

Antibody production

The invention provides antibodies that specifically bind to human IΡN-γ. These antibodies can be obtained by immunization with full-sized IΡN-γ or its fragments. Antibodies of the invention may be polyclonal or monoclonal and / or may be recombinant antibodies. In some embodiments, fully human antibodies of the invention are obtained, for example, by immunizing transgenic animals capable of producing human antibodies (see, for example, international patent application, publication No. U 93/12227).

SECs of the light chain and the variable regions of the heavy chain of the anti-IΡN-γ antibodies of the invention can be transplanted into frame sections (PK) from the same or different species. In some embodiments of the SEC of the light chain and variable regions of the heavy chain, anti-IΡN-γ antibodies can be transplanted into consensus human PKs to create a humanized antibody. Such humanized antibodies are encompassed by the present invention. To create consensus human RKs, PKs from several human heavy chain or light chain amino acid sequences are compared to identify a consensus amino acid sequence. RK heavy chain or light chain anti-IΡN-γ antibodies can be replaced by RK from another heavy chain or light chain. The rare amino acids in the PK of the heavy and light chains of the anti-IΡN-γ antibody are usually not substituted, while the remaining amino acids in the PK can be substituted. Rare amino acids are specific amino acids that are located at positions in which they are usually not located in the RK. The transplanted variable regions of the anti-IΡN-γ antibodies of the invention can be used with a constant region that is different from the original constant region of the anti-IΡN-γ antibody. Or, the transplanted variable regions are part of a single chain Ρν antibody. SEC transplantation is described, for example, in patents I8 6180370, 5693762, 5693761, 5585089 and 5530101, which are incorporated herein by reference for any purpose.

Antibodies of the invention can be obtained using transgenic mice having a significant portion of the human antibody-producing locus embedded in the antibody-producing mouse cells, and which are further modified by genetic engineering so as to be defective in the production of endogenous murine antibodies. Such mice are capable of producing human immunoglobulin molecules and antibodies and do not produce or produce substantially reduced amounts of murine immunoglobulin molecules and antibodies. The technologies used to achieve this result are described in the patents, applications and references disclosed herein in the description. In some embodiments, a person skilled in the art may use the methods disclosed in International Patent Application Publication No. U0 98/24893, which is incorporated herein by reference for any purpose. See also the article άеηάеζ е1 а1., 1997, No. 1 igene Senes ^ 11: 146-156, which is incorporated herein by reference for any purpose.

The monoclonal antibodies (mAbs) of the invention can be prepared in a variety of ways, including the standard methodology for producing monoclonal antibodies, for example, the standard method for hybridizing somatic cells according to ΚοΙιΡγ and Μίΐδ ^ ίη (1975, No. 1ite 256: 495). Although somatic cell hybridization procedures are preferred, other principles may in principle be used.

- 23 011876 methods for producing monoclonal antibodies, for example, viral or oncogenic transformation of Vlymphocytes.

One of the possible animal systems for producing hybridomas is a mouse. The production of hybridomas in mice is conventional, and immunization protocols and methods for isolating immunized splenocytes for fusion are well known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion techniques are also known.

In some embodiments, fully human monoclonal antibodies against ΙΡΝ-γ, possibly human ΙΡΝ-γ, can be obtained using transgenic mice carrying parts of the human immune system, and not the mouse system. These transgenic mice, referred to herein as HIMA mice, contain a mini-locus of human immunoglobulin genes that encodes the ungrouped sequences of the heavy (μ- and γ-) and k-light chains of human immunoglobulins along with predetermined mutations that inactivate the endogenous μ- and -chains (Lobberd! a1., 1994, No. 368: 856-859). Correspondingly, mice have reduced expression of murine ΙμΜ or k-, and, in response to immunization, the transgenes of human heavy chains and light chains introduced undergo class switching and somatic mutation with the formation of high-affinity k-monoclonal Ιμ человека human antibodies (Lebende! A1., Above; Lebdegde apy Nikkhag, 1995, Shet. Ket. Ittipo 1. 13: 65-93; Nagishd apy Lopegd, 1995, App. Ν.Υ. Asai. 8c1. 764: 536-546). The preparation of HIMB mice is described in detail in Tau1og e! A1., 1992, No. 1s1e1s Like Kek. 20: 6287-6295; Snep e! A1., 1993, Ip! etΐ ^ opa1 Iptipodod 5: 647-656; TiaShop e! A1., 1994, 1. Shtipo1. 152: 2912-2920; B eb e e! A1., 1994, No. 11ige 368: 856-859; Lobbegd, 1994, Naplob o £ Exp. P11agtasodod 113: 49-101; Tau1og e! A1., 1994, Shegpa! jupa1 Shtipo ^ du 6: 579-591; Ljöbgd & Nikhag 1995, Shet. Ket. Shtipo 1. 13: 65-93: Nagishd & Löbgd, 1995, App. KU. Asai. δα 764: 536-546; ЕЫ1 \\ З1й е! A1., 1996, Book Bio! espododu 14: 845-851, the contents of all of them are hereby incorporated by reference in their entirety. See further patents I8 5545806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5874299 and 5770429; all Lobbegd and Kau, as well as U8 patent 5545807 8gash et al., publication of international patent application U0 93/1227, published June 24, 1993; Y0 92/22646 published December 23, 1992; and \ U0 92/03918, published March 19, 1992, all disclosures of which are hereby incorporated by reference in their entirety. Alternatively, the strains of transgenic mice HCO 7 and HCO 12 described in the examples below can be used to obtain human anti-IΡN-γ antibodies.

In these embodiments, the antibodies of the invention specifically bind to ΙΡΝ-γ with an equilibrium dissociation constant (K _s ) of less than 10 ^-7 , 10 ^-8 , 10 ^-9 or 10 ^-10 M. In some embodiments of the invention, the antibodies bind to ΙΕΝ-γ with K _{with a} component of from about 10 ^-8 to 10 ^-12 M.

In preferred embodiments, the antibodies of the invention are a Ι§Ο1-, Ι§Ο2- or C ^ -isotype. Antibodies can be an Id ^ 1 isotype. In other embodiments, the antibodies of the invention are a Ι§Μ-, ΙβΛ-, CE- or CE-isotype. In preferred embodiments of the invention, the antibodies comprise a human kappa light chain and human CO 1 heavy chain. The expression of antibodies of the invention, including the constant region of the heavy chain Ι§Ο1, is described below in the examples. In specific embodiments, the variable regions of the antibodies are ligated with a constant region different from the constant region of the Id ^ 1 isotype. In some embodiments, the antibodies of the invention have been cloned for expression in mammalian cells.

In some embodiments, conservative modifications in the heavy chains and light chains of anti-γ antibodies (and corresponding modifications in coding nucleotides) will produce anti-γ antibodies having functional and chemical characteristics similar to those for anti-γ antibodies. In contrast, significant modifications in the functional and / or chemical characteristics of anti-ΙΡΝ-γ antibodies can be made by selecting substitutions in the amino acid sequence of the heavy and light chains, which differ significantly in their effect on maintaining: (a) the structure of the molecular skeleton in the region replacement, for example, with β-layer or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the side chain.

For example, a conservative amino acid substitution may mean replacing the native amino acid residue with a non-native residue, so that there is little effect on the polarity or charge of the amino acid residue in this position or no effect occurs. Moreover, any native residue in the polypeptide can also be substituted with alanine, as previously described for alanine scanning mutagenesis.

The desired amino acid substitutions (conservative or non-conservative) may be determined by those skilled in the art when such substitutions are desired. In some embodiments, amino acid substitutions can be used to identify important anti-γ antibody residues or to increase or decrease the affinity of the anti-γ antibodies described herein.

In alternative embodiments, the antibodies of the invention may express other cell lines besides hybridoma cell lines. In these embodiments, sequences encoding specific antibodies can be used to transform a suitable host cell host

- 24 011876 accumulating. According to these embodiments, the transformation can be accomplished by any known method of introducing polynucleotides into a host cell, including, for example, packaging the polynucleotide into a virus (or into a viral vector) and transducing the host cell by a virus (or vector), or using transfection techniques known in this areas as disclosed in the examples in patents I8 4399216, 4912040, 4740461 and 4959455 (which are thus all incorporated herein by reference for any purpose). A commonly used transformation technique may depend on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to, dextran-mediated transfection, precipitation of calcium phosphate, polybrin-mediated transfection, protoplast fusion, electroporation, encapsulation of a polynucleotide (polynucleotide) into a liposome and into the core.

Nucleic acid molecules (or polynucleotides) encoding the amino acid sequence of the constant region of the heavy chain, the variable region of the heavy chain, the constant region of the light chain or the variable region of the light chain of an anti-γ antibody of the invention are within the scope of the present invention. Such polynucleotides can be inserted into a suitable expression vector by standard ligation methods. In a preferred embodiment, a polynucleotide encoding the constant region of a heavy chain or light chain of an anti-γ antibody is attached to the right of the end of the polynucleotide encoding the corresponding variable region and are ligated into an expression vector. The vector is usually selected so that it is functional in the particular host cell used (i.e., the vector must be compatible with the apparatus of the host cell, so that this gene can be amplified and / or expressed). For a review of expression vectors, see METH. ΕΝΖ. 185 (6oebbe1, eb.), 1990, Asabetk Rge55.

Typically, expression vectors used in any of the host cells will contain sequences for maintaining the plasmid and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as flanking sequences, in some embodiments will typically contain one or more of the following nucleotide sequences: promoter, one or more enhancer sequences, a replication initiation point (opt), a transcription termination sequence, a complete intron sequence containing a donor and an acceptor splice site, sequence encoding a leader sequence for secretion of the polypeptide, ribosome binding site, after a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element. Each of these sequences is discussed below.

Perhaps the vector may contain! Hell '' - the coding sequence (! Ad-label), i.e. an oligonucleotide molecule located at the 5'- or 3'-end of the sequence encoding the anti-ΙΕΝ-γ antibody polypeptide; moreover, this oligonucleotide sequence encodes polyH15 (for example, hexaH1 ₈ ), or other! ad, for example EAC, HA (influenza virus hemagglutinin), or a tus for which antibodies are commercially available. This 1ad is usually associated with the polypeptide after its expression and can serve as a means for affinity purification or detection of ΙΕΝ-γ antibodies from the host cell. Affinity purification can be carried out, for example, using column chromatography using antibodies against β ad as an affinity matrix, β ad can then be removed from the purified anti-γ-antibody polypeptide in various ways, for example, using specific peptidases for cleavage.

Flanking sequences can be homologous (i.e., from the same species and / or strain as the host cell), heterologous (i.e., from a species different from the species or strain of the host cell), hybrid (a combination of flanking sequences from more than one source), synthesized or native. As such, the source of the flanking sequence can be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence is functional in the apparatus of the host cell and can be activated in it.

Flanking sequences useful in the vectors of this invention can be obtained by any of several methods well known in the art. Typically, flanking sequences useful for carrying out the invention will be pre-identified by mapping and / or digestion with restriction endonucleases and, thus, can be isolated from a suitable tissue source using suitable restriction endonucleases. In some cases, the complete nucleotide sequence of the flanking sequence may be known. In this invention, a flanking sequence can be synthesized using the methods described herein for the synthesis or cloning of nucleic acids.

Regardless of whether all or only part of the flanking sequence is known, it can be obtained by polymerase chain reaction (PCR) and / or by screening

- 25 011876 library using a suitable probe, such as an oligonucleotide and / or a fragment of a flanking sequence from the same or another species. If the flanking sequence is not known, then the DNA fragment containing the flanking sequence can be isolated from a larger DNA fragment, which may contain, for example, a coding sequence or even another gene or genes. Isolation can be performed by digestion with restriction endonucleases to obtain the corresponding DNA fragment, then isolation using agarose gel purification, column chromatography O | adep * (SBA! K ^ og1b, CA) or other methods known to the skilled person. The selection of suitable enzymes for this task will be apparent to one of ordinary skill in the art.

The origin of replication is usually part of those prokaryotic expression vectors that are commercially available, and this initiation point contributes to the amplification of the vector in the host cell. If the selected vector does not contain a replication initiation site, then it can be chemically synthesized based on a known sequence and ligated into a vector. For example, the site of initiation of replication from plasmid pBB322 (Νον Ε ^^ ά Вю1ак, Веуег1у, МА) is suitable for most gram-negative bacteria, and various viral initiation sites (for example, δν40, poliomavirus, adenovirus, vesicular stomatitis virus (νδν), or papillomavirus as Η₽ν or ASU) are useful for cloning vectors in mammalian cells. Typically, a replication component initiation site is not required for mammalian expression vectors (for example, the δν40 initiation site is often used only because it also contains an early promoter of this virus).

The transcription termination sequence is typically located at the 3'-end of the polypeptide-coding region and serves to terminate the transcription. Typically, the transcription termination sequence in prokaryotic cells is an O-C-rich fragment followed by a poly-T sequence. Although this sequence is easily cloned from a library or even commercially available as part of a vector, it can also be easily synthesized using nucleic acid synthesis methods, such as the methods described herein.

A selectable marker gene encodes a protein necessary for the survival and growth of a host cell growing in a selective culture medium. Typical breeding marker genes encode proteins that: (a) give prokaryotic host cells resistance to antibiotics or other toxins, such as ampicillin, tetracycline or kanamycin; (b) make up for auxotrophic cell deficiencies; or (c) provide critical nutrients not available from a complex or predetermined environment. Preferred breeding markers are the kanamycin resistance gene, ampicillin resistance gene and tetracycline resistance gene. Advantageously, the neomycin resistance gene can also be used for selection in both prokaryotic and eukaryotic host cells.

Other breeding genes may be used to amplify the gene to be expressed. Amplification is a process in which the genes required to produce the protein necessary for growth or cell survival are repeated in tandem within the chromosomes in subsequent generations of recombinant cells. Examples of suitable breeding markers for mammalian cells include dihydrofolate reductase (b1bubo о o1a! Hebis1ake, ΌΗΕΕ) and non-motor thymidine kinase genes. Mammalian transformant cells are placed under selection pressure, where only the transformants are uniquely adapted to survive due to the selectable gene present in the vector. The selection pressure is applied by culturing the transformed cells under conditions in which the concentration of the selective agent in the medium is gradually increased, thereby leading to amplification of both the breeding gene and the DNA that encodes another gene, for example, an antibody capable of binding to the ΙΕΝ-γ polypeptide. As a result of this, increased amounts of a polypeptide, such as an anti-γ antibody, are synthesized from amplified DNA.

The ribosome binding site is usually necessary to initiate mRNA translation and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is usually located 3 'from the promoter and 5' from the coding polypeptide sequence to be expressed.

In some cases, for example when glycosylation is desired in the expression system of a eukaryotic host cell, various pre- or pro-sequences can be applied to improve glycosylation or yield. For example, you can modify the peptidase cleavage site for a particular signal peptide or add pro-sequences that can also affect glycosylation. The final protein product may have at position -1 (relative to the first amino acid of the mature protein) one or more additional amino acids characteristic of expression, which may have not been completely removed. For example, the final protein product may have one or two amino acid residues found at the peptidase cleavage site attached to the amino terminus. Or, the use of certain enzyme cleavage sites may give a slightly truncated form of the desired polypeptide if the enzyme cleaves in such an area

- 26 011876 inside the mature polypeptide.

The expression and cloning vectors of the invention will typically contain a promoter that is recognized by the host organism and is operably linked to a molecule encoding an anti-IΡN-γ antibody. Promoters are non-transcribed sequences located in the opposite direction (i.e., in the 3A5 ′ direction) from the start codon of the structural gene (usually in the range of about 100 to 1000 bp), which control the transcription of the structural gene. Promoters are traditionally divided into two classes: inducible promoters and constitutive promoters. Inducible promoters initiate elevated levels of transcription with DNA under their control in response to some change in cultivation conditions, for example the presence or absence of a nutrient or a change in temperature. On the contrary, constitutive promoters constantly transcribe the gene with which they are functionally linked, i.e. with little control during gene expression or no control at all. A large number of promoters recognized by various possible host cells are well known. A suitable promoter is operably linked to DNA encoding a heavy chain or light chain comprising an anti-IBN-γ antibody of the invention by removing the promoter from the original DNA by restriction enzyme digestion and inserting the sequence of the desired promoter into the vector.

Suitable promoters for use in yeast hosts are also well known in the art. Preferably, yeast enhancers are used with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, promoters derived from virus genomes such as polyomavirus, avian pox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus , cytomegalovirus, retroviruses, hepatitis B virus, and most preferably monkey virus 40 (8U40). Other suitable mammalian promoters include heterologous mammalian promoters, for example, heat shock promoters and the actin promoter.

Additional promoters that may be of interest include, but are not limited to, the early promoter 8U40 (Vepo1k1 apb Sbatop, 1981, No. 290: 304-10); SMU promoter (TNotkep e! a1., 1984, Proc. No.! 1. Asab. I8A 81: 659-663); the promoter contained in the 3'-long terminal repeat of Routh sarcoma virus (Huatato, e! a1., 1980, Ce11 22: 787-97); the thymidine kinase promoter of the herpes virus (^ adpeg e! a1., 1981, Propos. No. 1. Asab. 8c1. I.8.A. 78: 1444-45); the promoter and regulatory sequences from the metallothionine gene (Vgshk! er e! a1., 1982, No.! ige 296: 39-42) and prokaryotic promoters such as the beta-lactamase promoter (Usha-KatagoGG e! a1., 1978, Proc. No. 11. Asab. Δα. I.8.A. 75: 3727-31); or! as promoter (EeBoeg e! a1., 1983, Proc. No. 111. Asab. δα. I.8.A. 80: 21-25). The following transcriptional control regions of animals that exhibit tissue specificity and have been used in transgenic animals are also of interest: the control region of the Σ elastase gene, which is active in pancreatic acinar cells (δ ^ ίή е! А1., 1984, Се11 38: 639-46; 0πάζ e! A1., 1986, Co1b δρπιΐβ Nagyog δutρ. Tsiap !. Vu1. 50: 399-409 (1986); MasOop1b, 1987, Nera! O1o§u 7: 425515); the control region of the insulin gene, which is active in pancreatic beta cells (Naiyap, 1985, No! IgE 315: 115-22); the control region of the immunoglobulin gene that is active in lymphoid cells (SokoksNeb1 e! a1., 1984, Ce11 38: 647-58; Abatek e! a1., 1985, No.! yoke 318: 53338; A1exapbeg e! a1., 1987, Mo1 . Ce11. Vu1. 7: 1436-44); the control region of the mouse mammary tumor virus, which is active in testicular cells, breast cells, lymphoid and mast cells (Lebeg e! a1., 1986, Ce11 45: 485-95); the control region of the albumin gene that is active in the liver (Rshkey e! a1., 1987, Sepek apb Eee1 1: 268-76); the control region of the alpha-fetoprotein gene, which is active in the liver (Kgishk-shG e! a1., 1985, Mo1. Ce11 Wu1. 5: 1639-48; Nutteg e! a1., 1987, δΑι ^ 235: 53-58); the control region of the alpha 1-antitrypsin gene, which is active in the liver (Ke1keu e! a1., 1987, Sepek and Eee1 1: 161-71); the control region of the beta-globin gene, which is active in myeloid cells (Modgat e! a1., 1985, No.! yoke 315: 338-40; KoShak e! a1., 1986, Ce11 46: 89-94); the control region of the myelin base protein gene, which is active in oligodendrocyte cells in the brain (YaeabBeab e! a1., 1987, Ce11 48: 703-12); the control region of the myosin gene of light chains-2, which is active in skeletal muscle (δηΐ'ΐί. 1985, No.! Ime 314: 283-86); and the control region of the gonadotropin-releasing hormone gene that is active in the hypothalamus (Makop e! a1., 1986, δαι'ΐΐίν 234: 1372-78).

An enhancer sequence can be inserted into a vector to enhance transcription of DNA encoding the light chain or heavy chain that make up the anti-IEN-γ antibody of the invention by higher eukaryotes. Enhancers are ak-acting DNA elements, usually about 10-300 bp. in length, which act on the promoter to enhance transcription. Enhancers are independent of orientation and position; they were detected both 5 'and 3' relative to the transcription unit. Several enhancer sequences are known that are accessible from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein and insulin). However, a virus enhancer is usually used. Enhancer δν40, enhancer of the early cytomegalovirus promoter, polyoma enhancer

- 27 011876 viruses and adenovirus enhancers known in the art are examples of enhancer elements for activating eukaryotic promoters. Although the enhancer can be located in a vector in either the 5 ′ or 3 ′ direction relative to the coding sequence, it is usually located at a site 5 ′ from the promoter.

A sequence encoding a suitable native or heterologous signal sequence (leader sequence or signal peptide) can be introduced into the expression vector to stimulate the secretion of antibodies from the cell. The choice of a signal peptide or leader sequence depends on the type of host cells in which the antibody is to be produced, and the heterologous signal sequence can replace the native signal sequence. Examples of signal peptides that are functional in mammalian host cells include the following: the signal sequence for interleukin-7 (1b-7) described in IB patent 4965195; the signal sequence for the interleukin-2 receptor described in Socapt e1 a1. (1984, HaYuge 312: 768); the signal peptide of the interleukin-4 receptor described in patent EP 0367566; the signal peptide of the receptor type I of interleukin-1 described in the patent IB 4968607; interleukin-1 type II receptor signal peptide described in EP 0 460 846; the human 1dK signal sequence (which is METTB ^ UB ^ URSBTS; BEO Yu XO: 52); the signal sequence of human growth hormone (which is ΜATSBΚTB ^^^ АΕС ^^ С ^ РV ^^ ЕББА; БЕО Ю ХО: 53) and the signal sequences of МБТАТАЬЬААУРУСУСУ (БЕО Ю ХО: 54) and METRA ^^^ Ε ^^^^ V ^ P ^ TTC (BEO Yu XO: 55) of a person encoded by cDNA encoding the heavy and light chains isolated in Example 3.

The expression vectors of the invention can be constructed based on a starting vector, for example, a commercially available vector. Such vectors may or may not contain all of the desired flanking sequences. If one or more flanking sequences described herein are not yet present in the vector, then they can be individually obtained and ligated into a vector. The methods used to obtain each flanking sequence are well known to those skilled in the art.

After the vector has been constructed and the nucleic acid molecule encoding the light chain, heavy chain or light and heavy chains constituting the anti-SHX antibody is inserted into the corresponding site of the vector, the completed vector can be introduced into a suitable host cell for amplification and / or expression of the polypeptide. Transformation of the anti-CX antibody expression vector into a selected host cell can be carried out by well-known methods, including transfection, infection, co-precipitation with calcium phosphate, electroporation, microinjection, lipofection, DEAE-dextran-mediated transfection, or other known methods. The method chosen will depend in part on the type of host cell used. These and other suitable methods are well known to those skilled in the art and are set forth, for example, in Batok e1 a1., Supra.

When cultured under suitable conditions, the host cell synthesizes an anti-CX antibody, which can then be assembled from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted). The selection of a suitable host cell will depend on various factors, such as the desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation), and ease of folding into a biologically active molecule.

Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese hamster ovary cells ( CHO, Sypeke Iatkeg ouatu), HeBa cells, hamster baby kidney cells (BHK, LaBe Iatkeg k1bpeu), monkey kidney cells (BSC), human hepatocellular carcinoma cells (for example, Hep C2) and many other cell lines. In some embodiments, cell lines can be selected by determining which cell lines have high expression levels and constitutively produce antibodies with ΣΕΚ-γ binding properties. In another embodiment, a cell line derived from B cells that does not produce its own antibody can be selected, but has the ability to produce and secrete a heterologous antibody.

Antibodies of the invention are useful for detecting CX-y in biological samples and for identifying cells or tissues that produce CX-y protein. Antibodies of the invention that specifically bind to SHX-y may be useful in the treatment of diseases mediated by SHX-y. These antibodies can be used in binding assays to determine SHX-y and to inhibit the formation of a complex of SH-y receptors of SH-y. These antibodies, which bind to SHX-y and block the interaction with other binding compounds, can find therapeutic application in the regulation of diseases mediated by SH-y. In preferred embodiments, antibodies to SHX-y can block the binding of XEX-y to its receptor, as a result of which the cascaded signaling induced by SHX-y can be disrupted.

In some embodiments of the invention, pharmaceutical compositions are provided comprising

- 28 011876 a therapeutically effective amount of one or more antibodies of the invention together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant. Preferably, acceptable substances for the preparation of the compositions are non-toxic to recipients at the dosages and concentrations used. In preferred embodiments, pharmaceutical compositions are provided comprising a therapeutically effective amount of anti-γ antibodies.

In some embodiments, formulations suitable for preparing the compositions are preferably non-toxic to recipients at the dosages and concentrations used.

In some embodiments, the pharmaceutical composition may contain substances for modifying, maintaining or maintaining, for example, pH, osmotic pressure, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition. In such embodiments, compounds suitable for preparing compositions include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrosulfite); buffers (such as borate, bicarbonate, Tris-HC1, citrates, phosphates or other organic acids); fillers (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); colorants, flavors and diluents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or moisturizing agents (such as pluronic (p1igoshs8), PEG, sorbitol esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal (! у1охара1)); stability enhancing agents (such as sucrose or sorbitol); tonicity agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol); delivery vehicles; thinners; excipients and / or pharmaceutical adjuvants. See ΡΕΜΙΝΟΤΟΝ'8 RANGE 8C! E \ CE8. 18 ^L EsNiop. (A.Ya.Seppago, eb.), 1990, Musk RiSiPd Sotrapu.

In some embodiments, the optimal pharmaceutical composition will be determined by a person skilled in the art depending, for example, on the intended route of administration, delivery method and desired dosage. See, for example, ΡΕΜΙΝΟΤΟΝ'8 PAYMENT 8 and 8, above. In some embodiments, such compositions may affect the physical condition, stability, rate of release of 1p1y0 and the rate of elimination of 1p1l0 antibodies of the invention.

In some embodiments, the primary carrier or carrier in the pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable carrier or base may be water for injection, physiological saline, or artificial cerebrospinal fluid, optionally supplemented with other substances conventional in parenteral compositions. Neutral buffered saline or saline mixed with serum albumin are further examples of carriers. In preferred embodiments, the pharmaceutical compositions comprise Tris buffer with a pH of about 7.0-8.5 or acetate buffer with a pH of about pH 4.0-5.5 and may further comprise sorbitol or a suitable substituent thereof. In some embodiments of the invention, anti-ΙΡΝ-γ antibody compositions can be prepared for storage by mixing the selected composition having the desired purity with possible agents for preparing the composition (Κ.ΕΜΙΝΟΤΟΝ'8 RNAJA and TARGISE, above) in the form of a lyophilized cake or aqueous solution. In addition, in some embodiments, the anti-ΙΡΝ-γ antibody product may be formulated as a lyophilisate with suitable excipients such as sucrose.

The pharmaceutical compositions of the invention may be selected for parenteral delivery. Alternatively, the compositions may be selected for inhalation or for delivery through the digestive tract, for example orally. The preparation of such pharmaceutically acceptable compositions is within the skill of a person skilled in the art.

The components for preparing the compositions are preferably presented in concentrations that are acceptable for the administration site. In some embodiments, buffers are used to maintain the physiological pH of the composition or a slightly lower pH, typically in the range of about 5 to about 8.

If parenteral administration is contemplated, therapeutic compositions for use in this invention may be provided in the form of a pyrogen-free, parenterally administered aqueous

- 29 011876 solution containing the desired anti-IΡN-γ antibody in a pharmaceutically acceptable carrier. A particularly suitable carrier for parenteral injection is sterile distilled water, in which the anti-IΡN-γ antibody is prepared in the form of a sterile, isotonic solution, properly stored. In some embodiments, the preparation may include preparing the desired molecule in the form of a preparation with an agent, such as injectable microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, which can provide controlled or sustained release of a product that may be delivered via depot injection. In some embodiments, hyaluronic acid having the effect of maintaining sustained release into the bloodstream may also be used. In some embodiments, implantable drug delivery devices may be used to administer the desired antibody molecule.

The pharmaceutical compositions of the invention may be formulated for inhalation. In these embodiments, anti-IΡN-γ antibodies are preferably prepared as a dry respirable powder. In preferred embodiments, solutions of the anti-IΡN-γ antibody for inhalation can also be prepared with a propellant for aerosol delivery. In some embodiments, the solutions may be nebulized. Therefore, methods for introducing into the lungs and methods for preparing preparations are further described in international patent application PCT / I894 / 001875, which is incorporated herein by reference and describes the delivery of chemically modified proteins to the lungs.

It is also contemplated that the compositions may be administered orally. Anti-IΡN-γ antibodies that are administered in this manner can be prepared with or without carriers commonly used in the preparation of solid dosage forms, such as tablets and capsules. In some embodiments, the capsule may be formulated to release the active portion of the composition at a location in the gastrointestinal tract where bioavailability is maximal and presystemic disruption is minimal. Additionally, agents may be included to facilitate absorption of the anti-IΡN-γ antibody. Thinners, flavoring agents, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants and binders may also be used.

The pharmaceutical composition of the invention is preferably provided for containing an effective amount of one or a plurality of anti-IΡN-γ antibodies mixed with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water or another suitable vehicle, a single dose of solution can be obtained. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; or binding agents, such as starch, gelatin or gum arabic; or lubricants, such as magnesium stearate, stearic acid or talc.

Other pharmaceutical compositions will be apparent to those skilled in the art, including anti-IΡN-γ antibody preparations with delayed or controlled delivery. Methods for preparing a variety of other delayed or controlled delivery agents, such as liposome carriers, biodegradable microparticles, or porous beads and depot injections, are also known to those skilled in the art. See, for example, international patent application PCT / and 893/00829. which is incorporated herein by reference and discloses the controlled release of porous polymer microparticles for the delivery of pharmaceutical compositions. Slow release formulations may include semipermeable polymer matrices in the form of formulated products, such as films or microcapsules. Slow release matrices may include polyesters, hydrogels, polylactides (as disclosed in I8 3773919 and European Patent Application Publication EP 058481, each of which is incorporated herein by reference), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (81 Dtai e! a1., 1983, B1oro1 teg 22: 547-556), poly- (2-hydroxyethylmethacrylate) (Laideg e! a1., 1981, 1. Wuther. Maeeg. Cent. 15: 167-277 and Laideg, 1982 , SIET. Teei. 12: 98-105), ethylene vinyl acetate (Laideg e! A1., Above) or poly-E (-) - 3-hydroxybutyric acid (European patent application publication EP 133988). Sustained release compositions may also include liposomes, which can be prepared by any of several methods known in the art. See, for example, Erk! Et e! A1., 1985, Proc. No.! 1. Asab. 8s1. I8A 82: 3688-3692; the publication of European patent applications EP 036676, EP 088046 and EP 143949, incorporated herein by reference.

The pharmaceutical compositions used for the administration of ίη νίνο are typically provided in the form of sterile preparations. Sterilization can be performed by filtration through sterile filtration membranes. When lyophilizing a composition, sterilization using this method can be carried out either before or after lyophilization and dilution. Compositions for parenteral administration may be stored in lyophilized form or in solution. Parenteral compositions are usually placed in a container having a sterilely closed inlet, for example, an intravenous solution bag or vial having a stopper punctured by an intravenous injection needle.

After the pharmaceutical composition is prepared in the form of a preparation, it can be stored in sterile vials in the form of a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. Such drugs can be stored either in a ready

- 30 011876 howl to use the form, or in the form (for example, lyophilized), which is bred before administration.

The invention also provides kits for preparing a unit dosage form for administration. Each kit according to the invention can contain both a first container containing dried protein and a second container containing an aqueous composition. In some embodiments of this invention, kits are provided containing single-chamber and multi-chamber pre-filled syringes (for example, syringes filled with liquid and syringes filled with lyophilisate (1 uzugtdez)).

A therapeutically effective amount of a used pharmaceutical composition containing an anti-IΕN-γ antibody will depend, for example, on the therapeutic context and goals. One of skill in the art will recognize that appropriate dosage levels for treatment will partially vary depending on the molecule released, on the indication that the anti-IΕN-γ antibody is used, on the route of administration and size (body weight, body surface or organ size) and / or condition (age and general health) of the patient. In some embodiments, the clinician may split the dose and modify the route of administration in order to obtain the optimal therapeutic effect. A typical dosage may vary from about 0.1 μg / kg to about 30 mg / kg or more, depending on the factors mentioned above. In preferred embodiments, the dosage may vary from 0.1 μg / kg to about 30 mg / kg, possibly from 1 μg / kg to about 30 mg / kg, or from 10 μg / kg to about 5 mg / kg.

The frequency of dosing will depend on the pharmacokinetic parameters of a particular anti-IΕN-γ antibody in the preparations used. Usually, the doctor prescribes the composition until a dosage is achieved that gives the desired effect. Therefore, for some time, the composition may be administered in a single dose, or in two or more doses (which may or may not contain the same amount of the desired molecule), or in the form of a continuous infusion using an implantable device or catheter. Further clarification of the appropriate dose is carried out by average specialists in this field by standard methods and is included in the amount of work that is standardly performed by them. Suitable dosages can be determined using appropriate dose-response testing data. In some embodiments, the antibodies of the invention can be administered to patients for an extended period of time. Continuous administration of an antibody of the invention minimizes an unfavorable immune or allergic response typically associated with antibodies that are obtained against a human antigen in a non-human animal, for example, an incompletely human antibody or a non-human antibody produced in non-human species. by man.

The introduction of the pharmaceutical composition is carried out according to known methods, for example, orally, by injection intravenously, intraperitoneally, intracerebrally (intraparenchymally), intracerebrospinal, intramuscular, intraocular, intraarterial, by injection into the portal vein or into the affected area; using slow release systems or implantable devices. In some embodiments, the compositions may be administered by bolus injection, either continuously by infusion or using an implantable device.

The composition may also be administered topically by implantation of a membrane, a porous substance or other suitable substance on which the desired molecule is absorbed or encapsulated. In some embodiments where an implantable device is used, this device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be accomplished by diffusion, a sustained release bolus, or continuous administration.

The use of pharmaceutical compositions with the anti-I антиN-γ antibody of the invention ex νίνο may also be desirable. In such cases, the cells, tissues or organs that were taken from the patient are exposed with pharmaceutical compositions containing an anti-IΕN-γ antibody, after which these cells, tissues and / or organs are implanted back to the patient.

In particular, anti-IΕN-γ antibodies can be delivered by implanting certain cells that have been genetically engineered using methods, such as the methods described herein, for expression and secretion of a polypeptide. In some embodiments, such cells may be animal or human cells and may be autologous, heterologous, or xenogenic. In some embodiments, the cells may be immortalized. In other embodiments, in order to reduce the likelihood of an immunological response, the cells can be encapsulated to prevent infiltration into surrounding tissues. In other embodiments, the encapsulated materials are typically biocompatible, semipermeable polymer shells or membranes that release the protein product (s) but prevent damage to these cells by the patient’s immune system or other undesirable factors from surrounding tissues.

- 31 011876

Examples

The following examples, including experiments and results obtained, are provided for illustrative purposes only and should not be interpreted as limiting the invention.

Example 1. Obtaining human protein ΙΕΝ-γ from CHO cells.

The full-size human ΙΕΝ-γ cDNA was amplified by PCR (under standard conditions) using cDNA from the human spleen MagaShop-Weabu (C1oShes11) as a template. This sequence was subcloned into the plasmid ρΌ8Βα2. ΌΗ10Β cells (Exxepius soybean) were transformed with the plasmid ρΌ8Κα.2. DNA was obtained using standard methods, and CHO cells were transfected using the calcium phosphate method (8res1a1u Meb1a, Sch.). A clone of a highly expressing cell line was used to produce a serum-free conditioned medium.

Conditioned for CHO cells, media containing human ΙΕΝ-γ (Ni-ΙΕΝ-γ) were concentrated, dialyzed and purified by several stages of chromatography. The first stage was C-HP (Rnagtash) chromatography using a standard gradient of No. 1C1 to separate highly glycosylated forms of Hn-ΙΕΝ-γ from non-glycosylated ones. The C-HP pool was then purified by chromatography with wheat germ agglutinin (Lactobacillus spec.). The purified material was separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Coomassie staining with blue and silver. The purified material had more than 95% purity, as determined by staining SDS-PAGE and Coomassie blue and silver. This material was also analyzed using the gel-thrombus method (limulus-amoebocyte lysate, Shtilik Luteosu! E Luka! E), which showed a low level of endotoxins. The identity of 1ш-ΙΕΝ-γ was confirmed by Western blotting using anti-LR-285 ΝΑ antibody from B & ук 8uk et al. The final protein concentration was determined by absorbance (A280) using a method with an extinction coefficient, where A ₂₈₀ / extinction coefficient = concentration in g / l (extinction coefficient = 0.66).

Example 2. Obtaining human monoclonal antibodies against ΙΕΝ-γ.

Transgenic mice NIMAb (from 1pc topos1op1 apbobu - human monoclonal antibody).

Fully human monoclonal antibodies to β-γ were obtained using strains of transgenic mice HCO7, HCO12 and HCO7 + HCO12, each of which expressed the genes of human antibodies. In each of these strains, the endogenous Kappa light chain gene of the mouse was homozygously destroyed, as described in C1ep e! a1. (1993, ΕΜΒΟ. 12: 811-820), and the endogenous mouse heavy chain gene was homozygously destroyed as described in Example 1 of the publication of international patent application νθ 01/09187 (incorporated by reference). Each strain carried a human kappa light chain transgene, KCo5, as described in Είκ1 ^ ί16 e! a1. (1996, YaShge Vu! Esipodu 14: 845-851). The HCO7 strain carries the HCO7 human heavy chain transgene, as described in I8 patents 5545806, 5625825 and 5545807 (incorporated by reference). The HCO12 strain carried the human heavy chain transgene HCO12, as described in Example 2 of publication νΟ 01/09187 (incorporated by reference). The HCO7 + HCO12 strain carried heavy chain transgenes of both HCO7 and HCO12 and was hemizygous for each transgene. All of these strains are referred to here as Mice.

Immunization NIMA.

In order to obtain fully human monoclonal antibodies to β-γ, NiMa mice were immunized with purified recombinant human β-γ having an origin from E. coli cells or CHO as an antigen. Conventional immunization regimens for NIMA mice are described in Leben e! a1. (1994, Laithe 368: 856-859; Είκ1 ^ ί16 e! A1., Supra, and in the publication of the international patent application νΟ 98/24884, the ideas of each of which are incorporated herein by reference). The mice were 6-16 weeks after the first antigen infusion. A purified recombinant preparation (25-100 μg) of β-γ antigen (for example, purified from transfected E. co11 cells or CHO expressing β-γ) was used to immunize NIMAb mice intraperitoneally (ip) or subcutaneously (s / c).

Immunization of transgenic NIMAb mice was carried out with antigen in Freund's complete adjuvant, administering two injections, followed by 2-4-week I / B immunization (up to 9 administrations) with antigen in Freund's incomplete adjuvant. Each antigen (human ΙΕΝ-γ produced either in E. soybean cells or in CHO cells) was immunized with several dozen mice. A total of 91 mice were immunized with ΙΕΝγ 91 strains of HCO7, HCO12 and HCO7 + HCO12. The immune response was monitored via retroorbital blood.

For the selection of NIMAb mice producing antibodies binding to γ-γ, the serum from immunized mice was analyzed using δδ, as described in 81, ν16 e! a1. higher. Briefly: microtiter plates were coated with purified recombinant ΙΕΝ-γ from CHO or E. soy cells at a concentration of 1-2 μl / ml in phosphate buffered saline (PB8) and incubated in an amount of 50 μl / well at 4 ° C overnight, then blocked 200 μl / well of 5% chicken serum in PB8 / Teteep (0.05%). Plasma dilutions from immunized ΙΕΝ-γ mice were added to each well and incubated for 1-2 hours at ambient temperature. The plates were washed with PB8 / T / HEP and then incubated.

- 32 011876 whether with goat anti-human Ec-specific polyclonal 1§C reagent conjugated to horseradish peroxidase (NKR) for 1 h at room temperature. After washing, the plates were developed using an ABT8 substrate (81dta С'Нет1с1 Со., 8ΐ. Лошк, МО, catalog number А-1888, 0.22 mg / ml) and analyzed spectrophotometrically by determining the optical density (0Ό) at wavelengths from 415 to 495 nm. Mice with sufficient titers of anti-human γ immunoglobulin were used to produce monoclonal antibodies, as described below.

Obtaining hybridomas producing human monoclonal antibodies to IΕN-γ.

Mice were prepared for the production of monoclonal antibodies by re-immunization with an antigen intravenously 2 days before sacrifice and then the spleen was removed. Mouse splenocytes were isolated from HIMB mice and fused to the mouse myeloma cell line using PEG and standard protocols. Typically, 10-20 fusions were performed for each antigen.

Briefly: unicellular suspensions of splenic lymphocytes from immunized mice were poured into one fourth of the number of non-secreting murine P3X63-Ad8.653 mouse myeloma cells (ATCC, SKB number 1580) with 50% PEG (8pcs). Cells were seeded at a concentration of approximately 1x10 ⁵ / well in flat-bottomed microtiter plates followed by approximately 2-week incubation in selective medium containing 10% fetal calf serum, 10% P388B1- (ATCC, SKB number T1B-63) conditioned medium, 3-5 % cloning factor for hybridomas 0K1OE ^ (UEK), partially purified supplement for growth medium for hybridomas, originating from the medium used for culturing a mouse macrophage-like cell line in BMEM (Meb1a! esE, catalog number SKL 10013, with a high concentration of hl glucose, L-glutamine and sodium pyruvate) plus 5 mM HEPE8, 0.055 mM 2-mercaptoethanol, 50 mg / ml gentamicin and 1x NAT (81dta, SKB catalog number P7185). After 1-2 weeks, the cells were cultured in a medium in which NAT was replaced by NT.

The resulting hybridomas were analyzed for the production of antigen-specific antibodies. Individual wells were analyzed using E8A (described above) for human anti-I -N-γ monoclonal ΙβΟ antibodies. In the event that rapid hybridoma growth was observed, the medium was usually analyzed after 10-14 days. Antibody-secreting hybridomas were reseeded, assayed again, and if they were still positive for human 1dO, then anti-IΕN-γ monoclonal antibodies were subcloned at least twice by restrictive dilution. Stable subclones were then cultured at ν 11th to obtain small amounts of antibody in a tissue culture medium for purification and characterization.

Selection of human monoclonal antibodies that bind to ΣΕΚυ.

The E8A assay described above was used to screen for hybridomas that showed positive reactivity with the EEKu immunogen. Hybridomas secreting a monoclonal antibody that binds with high avidity to IEn-γ were subcloned and then characterized. One clone from each hybridoma, which retained the reactivity of the parent cells (as determined using E8A), was selected to create a cell bank of 5-10 vials stored in liquid nitrogen.

To determine the isotype of the monoclonal antibodies produced as described herein, an isotype-specific E8A was performed. In these experiments, the wells of the microtiter plates were coated with 50 μl / well of solution 1 with a concentration of 1 mg / ml of the anti-human Kappa light chain of the mouse in PB8 and incubated at 4 ° C. overnight. After blocking with 5% chicken serum, the supernatant from each monoclonal antibody tested and purified isotype control was applied to the plates. The plates were incubated at ambient temperature for 1-2 hours. The wells were then reacted with either human 1dO1 or 1dO ₃ specific horseradish peroxidase conjugated goat anti-human polyclonal antiserum and the plates were developed and analyzed as described above.

Monoclonal antibodies purified from hybridoma supernatants that showed significant binding to IEN-γ as determined by E8A were further tested for biological activity using a variety of bioassays, as described below. Selected antibodies were designated as 1119, 1121, 1118 *, 1121 * and 1118.

Example 3. Cloning of the heavy and light chains of anti-IEN-γ antibodies.

Hybridomas expressing INN-γ-binding monoclonal antibodies 1119, 1121, 1118 *, 1121 * and 1118, identified in Example 2 above, were used as sources to isolate total RNA using TKoG® reagent (1Nygodep), a single-phase phenol solution and guanidine isothiocyanate, suitable for isolation of total RNA, DNA and protein. The first cDNA strand was synthesized using a random primer with an extended adapter (5'-ООС СОО АТА ООС СТС CAN NNN NN4-3 ') (8ЕО ΙΌ N0: 23), and 5'-CACE (with fast amplification of cDNA ends); preparative analysis was performed using OEZHKASEK ™ Ky (1 # Yogodep), a kit for fast amplification of cDNA ends (CACE) with improved efficiency according to the manufacturer's instructions. To obtain the full cDNA encoding the light chain, a forward primer was used, which was an ONEKASEC ™ nested primer, and the reverse primer was a 5'-OTS AoO STO OAA STO AOO-3 '(8ЕО ΙΌ N0: 24). The reverse primer was designed to recognize a conserved region of the sequence

- 33 011876 cDNA located in the 3'-untranslated region of the human kappa chain. Upon receipt of the cDNA encoding the variable region of the heavy chains, the forward primer was a SEKHEVASEV ™ nesting primer, and the reverse primer was 5'-TCA CCA CCC TCA CCA CAC C-3 '(8E0 ΙΌ NO: 25), which was designed to be recognized as conservative regions in the coding sequence in the Ec region of human C chains. BACE products were cloned into pCB4-TOPO (Ιπνί! Godep) and sequences were determined. Consensus sequences were used to design primers for PCR amplification of the full-length antibody chain.

To obtain a cDNA encoding an anti-β-γ kappa light chain, the 5'-PCR primer encoded the amino terminus of the signal sequence, the restriction enzyme XLaI site, and the optimized Kozak sequence (5'-ACA ACA AAC CTT STA CAC CAC CCA CCA AAC CCC ACC TSA SST TST STT-3 '; 8ЕО Ш NO: 26). The 3'-Primer encoded the carboxyl terminus and the termination codon, as well as the restriction site for 8aI (5'-CTT STS CAC TCA ACA STC TCC CCT CTT CAA CCT-3 '; 8EO ΙΌ 27). The resulting PCR product fragment was purified, digested with XBaE and 8aL, and then the gel was separated and ligated into the mammalian expression vector pE8Ba19 (see international application publication UO 90/14363, which is hereby incorporated by reference for any purpose).

To obtain the cDNA encoding the anti-β-γ heavy chain, a 5'-PCR primer was used that encoded the amino terminus of the signal sequence, the restriction site Xb! and the optimized Kozak sequence (5'-CAC CAC AAS CTT STA CAC CAC SAT CCC CTC AAC CCC CAT CCT-3 '; 8EO ΙΌ NO: 28). The 3'-Primer encoded the carboxyl terminus of the variable region, including the natural site for Hcts in the sense strand (5'-CTT CCT CCA CCC AST ACA CAC CCT CAC CAC CCC CCC CCC-3 '; 8EO ΙΌ NO: 29). The resulting product was purified, digested with XbAE, and BctBE gel was separated and ligated into the pO8Ba19 vector containing the constant region of human alkali.

Example 4. Expression of anti-ΙΕΝ-γ antibodies in Chinese hamster ovary cells (CHO).

Stable expression of anti-ΙΕΝ-γ mAb 1119 was achieved by cotransfection of plasmids containing the 1119 / pO8Ba19 heavy chain and 1119 / pO8Ba19 kappa chain into dihydrofolate reductase (ENE ^- ) defective serum-free Chinese hamster ovary cells (CHO). Transfected cells were selected in medium containing dialyzed serum but not containing hypoxanthine thymidine to ensure the growth of cells expressing the ENE enzyme. Transfected clones were analyzed using assays such as E8A to determine the expression of anti-γ-MAT 1119 in conditioned medium. Cell lines expressing 1119 were amplified in the presence of methotrexate. High amplification clones after amplification were selected to clone individual cells and create cell banks.

Any recombinant anti-ΙΕΝ-γ antibody of the invention can be obtained in Chinese hamster ovary cells defective according to ENE using the same protocol as described above for mAb 1119. DNA sequences encoding the full heavy chain or light chain of each anti-ΙΕΝ- γ antibodies of the invention are cloned into expression vectors. CHO cells defective in ENE are co-transfected with an expression vector capable of expressing the full heavy chain and an expression vector expressing the full light chain of the corresponding anti-γ antibody. For example, to obtain antibody 1118, cells are co-transfected with a vector capable of expressing the complete heavy chain comprising the amino acid sequence represented by 8EO N NO: 19 and a vector capable of expressing the complete light chain comprising the amino acid sequence represented by 8EO N NO: 20. To obtain antibody 1121, cells are transfected together with a vector capable of expressing the complete heavy chain, including the amino acid sequence shown in 8EO ΙΌ NO: 21, and the vector, it is desirable to express a complete light chain including the amino acid sequence shown in 8EO W NO: 22. To obtain the 1118 * antibody, cells are transfected together with a vector capable of expressing the full heavy chain including the amino acid sequence represented in 8EO ΙΌ NO: 32 and a vector capable of to express the complete light chain, including the amino acid sequence shown in 8EO ΙΌ NO: 20. To obtain antibody 1121 * cells are transfected together with a vector capable of expressing complete w heavy chain comprising the amino acid sequence as defined in the W 8EO NO: 21 and a vector capable of expressing a complete light chain comprising the amino acid sequence shown in 8EO ΙΌ NO: 33. Table. Figure 3 shows the full heavy and full light chains for different antibodies against ΙΕΝ-γ.

- 34 011876

Table 3

Antibody The variable region of the heavy chain + Heavy chain constant region Full heavy chain 1119 ZER GO N0: 6 + § £ (2 Yu N0: 2 ZER GO N0: 17 1118 ZER Yu N0: 10 + ZER GO N0: 2 ZER GO N0: 19 1121 ZER GO N0: 14 + ZER GO N0: 2 ZER GO N0: 21 1121 * ZER GO N0: 14+ ZER GO N0: 2 ZER GO N0: 21 1118 * 5Е0 GO N0: 30 + ZER GO N0: 2 ZER Yu N0: 32 Antibody Light chain variable region + Constant region light chains Full light chain 1119 ZER GO N0: 8 + ZER GO N0: 4 ZER GO N0: 18 1118 ZER GO N0: 12 + ZER GO N0: 4 ZER Yu N0: 20 1121 ZER GO N0: 16 + ZER GO N0: 4 ZER GO N0: 22 1121 * ZER GO N0: 31 + ZER GO N0: 4 ZER GO N0: 33

Antibody The variable region of the heavy chain + Heavy chain constant region Full heavy chain 1118 * ZER GO N0: 12 + ZER GO N0: 4 ZER GO N0: 20

Example 5. The production of anti-ΙΕΝ-γ antibodies.

Antibody 1119 was obtained by expression in a cloned cell line of CHO that expressed it. To start production, cells from one vial were thawed in a serum-free cell culture medium. Cells were grown first in a 250 ml shake flask, then in rotating flasks and, finally, in stainless steel reactors with increasing size up to a 2000 liter bioreactor. Production was carried out in a 2000 L bioreactor, working with a culture with the addition of recharge, where nutritional supplementation containing concentrated medium components is added to maintain cell growth and culture viability. Production was continued for approximately 2 weeks, during which cells constitutively produced antibody 1119 and secreted it into the culture medium.

In an industrial reactor, predetermined pH, temperature, and dissolved oxygen were monitored. The pH was controlled by the addition of carbon dioxide and sodium carbonate. Dissolved oxygen was controlled by flows of air, nitrogen, and gaseous oxygen.

At the end of production, the cell broth was fed into a centrifuge with conical baffles in the rotor (FKK! Ask) and the culture supernatant was separated from the cells. The concentrate was further clarified through a volumetric filter, then through a 0.2 μm filter. The clarified conditioned media was then concentrated by flow ultrafiltration along the stream. Conditioned media were concentrated 15-30 times. The resulting concentrated conditioned medium was then processed to purify the antibody contained therein, but it could be frozen for purification at a later stage. Any other antibody described herein can be obtained in a similar manner.

Example 6. Characterization of the activity of anti-ΙΕΝ-γ antibodies.

Since ΙΕΝ-γ has a large number of biological effects, several different bioassays were used to compare the activity of different antibodies to ΙΕΝ-γ. The A549 analysis described below was used for initial screening, where candidates for further analysis were selected according to their function.

- 35 011876 ni in this analysis. Selected candidates included antibodies 1119, 1118 and 1121.

Bioanalysis A549.

One of the established properties of ΙΕΝ-γ is its antiproliferative effect on various cell populations. See, for example, Aipe apb Rodie, 1989, 1. Cl. 1pte51. 84: 863-75. The human lung cell line A549 has often been used in publications to describe the актив-γ bioactivity. See, for example, Aipe Apb Rodie, above; H111 e! A1., 1993, ITTInodod 79: 236-40. In general: inhibitor activity is tested at a concentration of a stimulant that is part of the dose response curve where a small dose change will result in a change in response. One of skill in the art will understand that if an excess dose of a stimulant is used, then a very large dose of inhibitor may be required to observe the response change. Commonly used concentrations for the stimulant are EC80 and EC90 (concentrations at which 80 or 90% of the maximum response is achieved, respectively).

The dose-response curve was constructed to determine the EC ₉₀ for the A549 line of lung carcinoma epithelial cells (~ 30 pM). In subsequent experiments, different concentrations of purified antibodies were mixed with a fixed dose of ΙΕΝ-γ (30 pM) and the ability of the antibodies to inhibit the biological activity of the antiproliferative effect of ΙΕΝ-γ was determined. The analysis was carried out for 5 days and proliferation was measured by determining the fluorescence generated by decreasing ALAMACVIEE ™ (AssiMeb Shkgpatsopak, Shs., Sysoado, PNpo18) - a dye used to detect cell growth of metabolically active ones, i.e. proliferating cells. See, for example, Be Rpe5 apb MykyakY, 1995, 1. Cl. Bb. Apa1u515 9 (2): 89-95; Ayteb e! A1., 1994, 1. Ittiηo1. Meyob5 170 (2): 211-24.

As shown in FIG. 9, antibody 1119 was the most active antibody with Ιί. ' ₅₀ (concentration at which 50% inhibition of the ΙΕΝ-γ effect was achieved) of 14 pM, then the most active antibodies were 1121 (46 pM) and 1118 (97 pM).

Bioanalysis HLA ΌΚ.

Another established property of ΙΕΝ-γ is its ability to upregulate the expression of genes of the main histocompatibility complex (MHC) of class Ι and class ΙΙ in different types of cells. This activity can be especially significant in the case of lupus nephritis (Uokouata e! A1., 1992, K1bpeu Sh !. 42: 755-63). The THP-1 human monocytic cell line has often been used in publications describing this biological activity of ΙΕΝ-γ. The dose-response curve was constructed to determine the EC80 for the specific THP-1 cell line used in this experiment (approximately 21 pM). In subsequent experiments, different concentrations of purified antibodies were mixed with a fixed dose of ΙΕΝ-γ (21 pM) and the ability of antibodies to neutralize or inhibit ΙΕΝ-γ-induced upregulation of HLA ΌΚ expression on the cell surface was determined. The analysis was carried out for 24 hours, and the measured endpoint was the average fluorescence intensity, as determined using the ΕAC8 analysis (method of two-parameter sorting of the cells labeled with iododeoxyuridine in the duct) to determine the binding of the ΕIΤC-labeled anti-HLA antibody to the cells.

As shown in FIG. 10, antibody 1119 was the most active antibody with Ιί. ' ₅₀ 14 pM, then 1121 (60 pM) and 1118 (86 pM) were more active.

Bioanalysis of whole blood.

Analysis of human whole blood was developed on the basis of published observations that ΙΕΝ-γ positively regulates the production of chemokine GR-10 in several different cell lines. This activity can be especially significant in the case of lupus nephritis (ΝηΓίιιηί е! А1., 2000, Su! Ocher 12: 1561-1565). Whole blood from several normal human donors was tested for the ability of ΙΕΝ-γ to increase production of No.-10. The dose-response curve of γ-γ response was constructed to determine the EC50 for individual donors. As expected, some variation was observed between donors. In general terms: donors were used, which apparently reproducibly demonstrated an EC _{50 of} 50-100 pM. Whole blood was mixed with a fixed concentration of γ-γ and different concentrations of antibodies, incubated for 18.5 hours, and then with the help of E8A, the levels of YP-10 were determined. Typical whole blood test results for two different donors are shown in FIG. 11. Ιί. ' ₅₀ of these two donors were 17 and 14 pM. To date, only one donor has been identified with spontaneously elevated levels of TP-10 in a whole blood test without the need for exogenous ΙΕΝ-γ. Anti-ΙΕΝ-γ antibodies were able to block this spontaneous production of ΙΗ-10, probably by blocking the endogenously produced ΙΕΝ-γ.

Biochemical analyzes.

Binding kinetics for several anti-γ antibodies were measured using B ^ soy analysis. Initial results suggested that the antibodies had abnormal conditions, which led to limitations for reliable measurements on B ^ SOKE ™ (Ryagtasha Vu ₈ en ₈ en AB Sogrog! Ju, Irr5a1a, 8 ^ ebep), an apparatus that uses surface plasmon resonance for binding measurements

- 36 011876 between molecules. Accordingly, an analysis of equilibrium binding was developed and used. A fixed amount of antibody was incubated with different concentrations of IΡN-γ for more than 5 hours to achieve equilibrium and then brought into contact with IΡN-γ coated beads for a very short time and the amount of free antibody that bound to the beads was measured in a KSHEHA ™ device (§Ar ^ άуηе ΙηδΙπιιικηΙδ Шс., Βοί ^, ΙΌ), apparatus for immunoassay based on fluorescence. The smallest equilibrium dissociation constant (approximately 24 pM) was obtained with antibody 1119.

Example 7. Cross-reactivity of species.

The antibodies described above were tested for their ability to neutralize or inhibit recombinant IΡN-γ proteins from several different species. Murine IΡN-γ protein was commercially available, while human IΡN-γ proteins, monkeys suñotó1di8 and chimpanzees, were cloned and expressed in standard mammalian expression systems, such as 293 human cells. All human IΡN-γ proteins, suηοtо1di8 and chimpanzees, were previously active described analysis A549, while murine protein was inactive in this analysis. Mouse protein was active in the KA ^ 264.7 cell line assay, which was essentially identical to the previously described A549 assay, with the exception of replacing the murine cell line. KA ^ 264.7 is a monocytic macrophage line of mouse cells and can be obtained, for example, from the American type culture collection. As shown in the table. 4, all three antibodies were able to neutralize human IΡN-γ and chimpanzees, although none of the three antibodies was able to neutralize or inhibit the biological activity of IΡN-γ from sunto1di8 or from a mouse.

Table 4

Antibody Man Shnmp. Soup. Mouse 1118 Yes Yes Not Not 1119 Yes Yes Not Not 1121 Yes Yes Not Not

Example 8. Identification of the epitope of anti-IΡN-γ antibodies.

Comparison of the amino acid sequences of mature human IΡN-γ and suηοтοi8 showed that there are 9 amino acid differences between them at positions 19, 20, 31, 34, 65, 77, 103, 110, and 126 in the human IΡN-γ sequence. The sequences ^ N-7 of humans and chimpanzees are disclosed in Tyakig and ΕηηάοΙΓί (1999), ΜοΡαιΕίΓ Ittiηο1οu 36: 1107-15. The sequence of ^ N-7 monkeys sugute1di8 is disclosed in Ta18it1 and 8a1a (1997), Ιη1. Agsy. A11 where Ittiηο1. 114 (3): 229-36; and the sequence ^ N-7 of the mouse is disclosed, for example, in the number No. 032363 of the catalog of the National Center for Biotechnological Information (NСΒI, Ка1юия1 СеШег Γογ Βίο ^ ΐιηοίοβ} 'IηΓο ^ та1 ^ οη). Site-directed mutagenesis using a commercially available kit was used to individually replace each of the divergent human amino acids in human β-γ with the corresponding amino acid from protein sunto1di8. Each substituted ERTS-y was called siRH, 'followed by a symbol for the amino acid used to replace the amino acid present in the human sequence and the position in the mature W ^ sequence in the person in which the replacement occurred. For example, yi [P ^ P19 denotes a variant of W ^, identical to W ^ in humans, with the exception of position 19, where aspartic acid replaces the histidine present in this position normally. Similar experiments were performed with W ^ in monkey suηοοοοдиди88 and with the replacement of each divergent amino acid with the amino acid present in ^ N-7 of humans. For example, the notation suηοIΕNγ ^ 103 denotes the variant W ^ y, identical to ^ N-7 of the monkey suηοtο1di8, with the exception of position 103, where leucine replaces serine, normally present in this position. See table 5 and 6. These mutant proteins were expressed in standard mammalian expression systems, such as 293 human cells. All mutant ^ N-7 proteins remained active as determined in assay A549. Antibody 1119 was tested for its ability to neutralize or inhibit the biological activity of various mutant W ^ y proteins, as determined using A549 bioassay. The ability of antibody 1119 to neutralize or inhibit the antiproliferative activity of W ^ in humans was determined by measuring fluorescence as described above, and this was used as a basis for comparing the ability of antibody 1119 to neutralize the activity of each of the different forms of W ^. For constructs that were based on ^ N-7 human, inhibition of ^ N-7 activity was measured in percent, where the maximum level of fluorescence observed in the presence of ^ N-7 human and 1119 antibody (due to a decrease in A ^ AΜAKΜ ^ υЕ ™ by proliferating cells ), was taken as 100%, and the maximum levels measured in the presence of each of the modified forms of ^ N-7 plus antibody 1119 were compared with this value. Alternatively, for constructs that were based on ^ N-7 monkeys su8note8, inhibition was evaluated qualitatively in points based on the observed fluorescence.

As presented in the table. 5, antibody 1119 was able to neutralize or inhibit the biological activity of human ΙΡΝ-γ and all substituted human ΙΡΝ-γ mutants, with the exception of 1π.ιΙΕΝ ', Ό19, and 1ιιιΙΡΝγΡ20. As in the previous example, ΙΡΝ-γ protein supoto1§§ was also not inhibited by antibody 1119. This analysis shows that residues 19 and 20 are especially important for the interaction between antibody 1119 and ΙΡΝ-γ and can serve as contact points for antibodies 1119 and ΙΡΝ- γ person.

Table 5

ΙΡΝ-γ % neutralization by antibody 1119 ΙΡΝ-γ person one hundred ΙΡΝ-γ Suponyu! oiv 0 1ιιιΙΕΝΙΕΝ-τΟ19 0 ΗΐϊΙΓΝ-γΡ20 0 ΗιιΙΡΝΙΡΝ-γΟ31 103 ΗαΙΡΝ-γΚ34 109 ΗιιΙΡΝ-γΚ.65 109 ΗυΙΡΝ-γΙ77 108 1ηιΙΡΝ-γ8103 102 ΙηιΙΓΝ-γνίΙΟ 103 1ηιΙΡΝ-γ1126 109

Tab. Figure 6 shows that an altered version of the ΙΡΝ-γ of the monkey supoto1di8, including substitutions making the sequence of ΙΡΝ-γ supoto1dig the corresponding human sequence at positions 19 and 20, was not neutralized by antibody 1119. However, variants of the ΙΡΝ-γ monkey of supoto1di8 having substitutions in the human sequence in positions 19, 20 and 65, or 19, 20 and 103, were neutralized by antibody 1119, as were variants containing other substitutions in addition to any of these substitutions.

Table 6

Design Antibody neutralization 1119 ΙΡΝγ person Yes soup ΙΡΝγ not soup ΙΡΝγΗ 19/520 not soup ΙΡΝγ865 not soup ΙΡΝγυ03 not soup ΙΡΝγ865 / υ03 not soup ΙΡΝγΗ 19/520/565 Yes soup ΙΡΝγΗ 19/820 / B103 Yes soup ΙΡΝγΗ 19/520 / B103 / 1110 Yes soup ΙΡΝγΗ 19/820/365 / B103 / 1110 Yes

Example 9. The biological activity of anti-ΙΡΝ-γ antibodies with the introduction of chimpanzees.

Antibody 1119 was administered to 2 chimpanzees at a dose of 20 mg / kg every week for 3 weeks. Blood was drawn from chimpanzees either two (-2) or one (-1) week before the administration of the antibody, and then blood was taken 2, 8, 15, 29 and 36 days after the first dose of the antibody. An analysis of the whole blood of chimpanzees was carried out on blood taken using essentially the same method as used in the analysis of human whole blood described in Example 6, the key difference being that the antibody was not added exogenously to whole blood, but in advance introduced chimpanzees 1n u1uo. ΙΡΝ-γ was added to the blood at various concentrations (0.01, 3.9 ng / ml or 1 μg / ml), blood was incubated at 37 ° C for 20-24 hours, and then the production of ΙΡ-10 was measured using E8A on granule base.

- 38 011876

As seen in FIG. 14 and 15, blood taken from animals prior to antibody administration responded to ^ N-7 with a concentration-dependent increase in GR-10 production. In contrast, blood taken after administration of the antibody did not respond to any of the tested concentrations of GEL-y by an increase in GR10 production. These data show that the antibody retained the ability to neutralize ^ N-7 even after administration of w νίνο. In addition, the amount of ^^ 7 added to these cultures significantly exceeded the endogenous levels of ^ N-7 in chimpanzees, which suggests that the introduced antibody will be able to neutralize endogenous ^ N-7 w νίνο.

It should be understood that the foregoing disclosure places particular emphasis on certain specific embodiments of the invention and that all modifications or changes equivalent to this are within the spirit and scope of the invention as defined in the appended claims. All references cited herein are hereby incorporated by reference in their entirety.

The list of sequences <110> dtdep, 1ps.

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Niapd, skip <120 Human neutralizing antibodies against ΙΕΝ-gamma as selective inhibitors of the ΙΕΝ-gamma <130> 01-1635-b pathway

<150> 60 / 419,057 <151> 2002-10-16 <150> I5 60 / 479,241 <151> 2003-06-17 <160> 57

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<210> one <211> 990 <212> lnc <213> noto 5ar1epz

dststassa <400>

adddsssagh ddgsshssss stddsassskh sssssaadad sasstsgddd ddsasadsdd sssgdddsgd ssgddthaad dasgasgtss ssdaassddg dasddsdgsd gddaastsad dsdsssgdas sadsddsdhd sasassgsss sddstdgsst asadsssTsa ddasGsgasg sssgsadsad sdgddgdass dgdsssgssa dsadsggddd sasssadass

120

180

240

- 39 011876

tasahsgds asdgdaahsa saadssads aassassadd hddasadaa adshdadsss 300 aaagshhddhd asaaaaskhsa sasahdsssa ssdhdsssad sasshdaash sshdddddddda 360 ssdhsadgsg HSSSHHSSS sssaaaasss aaddasasss hsahdahsgs ssddasssskh 420 daddhsasah dsddhddhddh ddasdhdads sasdaadass shadddhsaa dhhsaaskhdd 480 hasdhddasd dsddhddddd £ dsahaasddss aadasaads sdsddddda dsadhasaas 540 adsasdhass dhddhddssad sdhsshsass dhsshdssass addashdddh daatdsdaad 600 dadhasaadh dsaaddhshs saasaadadss hssssadsss ssatsdadaa aassachshss 660 aaadssaaad ddsadssessd adaassasad dhdhasasss Hdssssss ssdddahdad 720 shdassaada assaddhsad ssgdasskhds shddhsaaad dskhskhagss sadsdasa 780 dssdhdddadh dddadadsaa Hdddsadssd dadaasaskh asaadassas dsskhsssddhd 840 schddashkhssd asdsdshhh schkhsskhsgat adsaads ssdhddasaa dadsadddhdd 900 kindergarten asdhshhhhs ahdskhssddhd ahdsahdadd sxhdsasaa ssashasasd 960 sadaadadss hkhsssshdhs ссдддддааааа 990

<210> 2 <211> 330

- _ ’P’P'T '<£! £> 11G I <213> Noto Zara'epz <400> 2

A1a Run thr love € 1y Rgo Run wah! Pb Rgo Bie A1a RGO Run Run love one 5 10 fifteen Run thr Run 61 y twenty 61U Tht l! a but! but yee 25 61 y suz Bie Wah! love thirty AZR Tug Pb Rgo 61and RGO wah! Tht wah! Run Tgr AZP Run C1u l! a yee thr Run 35 40 45 61 y wah! fifty ΗΊ 5 Tht Pb Rgo A1 a 55 wah! yee 61π Run Run 60 C1u yee tug Run this Zeg Run Wah! Wah! Thr wah! Rgo Run Run Run Bie 61y tkg 61 η thr 65 70 75 80 Tug 11th Suz AZP wah! 85 AZP ΗΊ5 love RGO Run 90 AZP Thr love wah! Azr 95 love ue Wah! 61 and RGO one hundred love Run suz Azr love 105 Thr H1 5 Thr Su 5 Rgo 110 Rgo Suz rgo but! but Rgo 61 and Bie Bie 61 y 61y rgo Run wah! Pb Bie Pb Rgo Rgo 115 120 125

- 40 011876

love RGO 130 Azr thr yee met 11e 135 Zeg Agde Thr Rgo 140 61 and wah! Thr SU 5 wah! wah! wah! Azr Wah! Zeg Oil refinery b! and A5r Rgo 61 and wah! love ₽Ke Azp Tgr 145 150 155 160 Tug ya1 AZR 61 y Ya1 61and wah! Ηίδ AZP A1a love thr love RGO Agd 61 and 165 170 175 C1 and 61 p Tug AZP Zeg Tht tug Agd Ya1 Wah! Zeg wah! yee Tht Wah! Bie 180 185 190 Ηίε 61 η AZR 195 Tgr yee AZP C1u love 200 61 and Tug love Suz ug 205 wah! zeg AZP love A 1a Bie Rgo A1a RGO 11th 61 and Bz Tht 11th zeg Bz A1a Bz 61 y 210 215 220 C1p Rgo Agd 61 and RGO 61 p ya1 Tug thr yee Rgo RGO zeg Agd A5r 61υ 225 230 235 240 her Tht Bz AZP 61p Ya1 Zeg Bie Tht suz Bie Ya1 love 61 y Pb Tug 245 250 255 Rgo 5eg A5r 11th A1a Ya1 61 and Tgr 61and Zeg AZP 61 y 61 η RGO 61 and AZP 260 265 270 AZP Tug Bz 275 Tht Tht Rgo RGO Ya1 280 Bie Azr zeg AZR 61 y 285 Zeg Pb Pb her Tug 290 Zeg Bz Bie Tht wah! 295 AZR love Zeg Agd tgr 300 61l 61 p 61y A5P Ya1 pke 5eg Suz Zeg wah! meth ΗΊ 5 61 and A1a Bie H13 AZP ΗΊ 3 tug thr 305 310 315 320 61 p Bz Zeg yee Zeg Bie zeg rgo 61 y

325 330 <21О> 3 <211> 321 <212> DNA <213> Noto Zartepz

<400> 3 sdaasttddd stdsassats tddsttsats TTsssdssah sdatdadsa dttdaaatst 60 ddaaskhdsst sttdttdttdd sstdstdaat aaststats ssadadadds saaadtasad 120 sdddaddtd ataasdssst ssaatsdddt aastsssadd adadtdtsas adadsaddas 180 adsaaddasa dsasstasad sssadsads assstdasds tddadsaads adastasdad 240 aaasasaad tstasdsssd sdaadtsass satsadddss TdadsTsdss sdtsasaad 300 adsttsaasa ddddadadtd ΐ 321

- 41 011876

<210> 4 <211> 107 <212> PRT <213> Noto Sartels

<400> 4

Agd TIG Wah! A1 a but! but Rgo 5eg Wah! Riye Not Pb Rgo Rgo Zeg AZR 61 and one 5 10 fifteen 61 η yee B5 Zeg C1u tyg but! but Zeg Wah! wah! Suz yee yee AZP AZP Rie twenty 25 thirty Tug RGO Agd 35 61 and Yes LU5. _ua1. 61 p 40 Tgr love Wah! A5r AZP 45 but! but yee 61 p Run 61y fifty AZP zeg C1p FROM! and Zeg 55 Wah! tyg from! and FROM! η Azr 60 Zeg love Azr Zeg Tyg 65 Tug 5eg Bie Zeg Zeg 70 Tyg yee tyg Bie zeg 75 love A1a Azr tug 61 and 80 Bz H13 Bw5 Wah! tug A1a suz 61 and wah! TIG ΗΊ5 61 p C1u Bie Zeg Zeg 85 90 95 RGO Wah! tyg Bz 5eg riye A5L Agd boo 61 and Su5 one hundred 105

<210> 5 <211> 351 <212> dna <213> note Zar ^ EPZ <400> 5

9 ^and 99 ^ 9sads hddhasadhs hddadsadad dhdaaaaads ssddddadhs hskhdaadahs hsskhdhaadd dhhskhddaha saaskhhtass adskhaskhdda hsddskhdddh dsdssadatd sssdddaaad dsskhddadhh dahddddahs ahstahsskhd dhdaskhskhda hassadahas adsssdhsskh hssaaddssa ddhsassahs hsadssdasa adhssahsad sassdsskhas skhdsadhdda dsadsskhdaa ddsskhsddas assdssahdh ahhaskhdhdd hhsddddads haskhhhhaskh hsdahskhskhd dddssdhdds assskhddtsa ssdhskhskhad x

120

180

240

300

351 <210> 6 <211> 117 <212> PRT

- 42 011876 <213> Noto Zaraepz <400> 6

61 and one ya1 61 p Bie νΗ1 5 61 η Zeg 61 y A1a 61 and 10 Ya1 Bw Bz RGO 61 y fifteen 61 and zeg yee Bw Not twenty Zeg Suz Bz b1u Zeg 25 61 y Tug AZP Pb Thr thirty zeg Tug tgr Pe 61 y 35 Tgr 7a1 Agd 61 η Meg 40 Rgo 61 y Bz 61 y yee 45 61 and yee fur 61 y Not fifty 11 e Tug RGO 61 y AZR 55 Zeg A $ p Thr Agd tug 60 zeg RGO Zeg Pb 61 p 65 61 y 61 p Ya1 tNg Not 70 Zeg D1a AZR 1-U5 Zeg 75 Not run Thr A1a Tug 80 yee 61 η Tgr zeg Zeg 85 yee bue A1a zeg Azr 90 Thr A1a fur Tug Tug 95 Suz 61 y 56G 61 y Zeg one hundred tug Pb Tug Pb Azr 105 bei Tgr 61 y Agd 61 y but Thr yee 7a1 Thr Ya1 115 Zeg Zeg <210> 7 <211> 324 <212> DNA <213> But that ZarTeps

400

180 dasaddhhsa dhdddsadhdd dkhskhdddasa dashkhsasts hsassahsad sadhashddad 240 sshdaadahh hhdsadhdha hhaskhdhsad sddhshdddhd dhsahsahh sashhhhddddh sdhhhhhhhhhhhhhhhhhhhh

324

<210> 8 <211> 108 <212> PRT <213> Noto Zartezz

- 43 011876 <400> 8

BT Not ya1 yee Thr 61 η Run RGO b1u Thr one 5 10 61 and Agd A1a Thr yee Run Suz DGD A1a Run twenty 25 tug Bie A1a 35 tgr Tug 61 p 61 η love 40 RGO 61 y Not tug b1u Yes Run Run Agd A1a tig 61 y fifty 55 61y 65 Run 61 y Run C1u Tht 70 AZR Pb thr yee Rgo 61 and AZR ₽Ke A1a Ya1 Tug .tug Suz C1p 85 90 pb Tht Rke C1u RGO 61y Tht love Ya1 Azr one hundred 105

Not <210>

Hei Run Hei Run Rgo fifteen 61 y 61 η Run UAZ Run Run Run thirty 61 η A1a RGO Agd hei hei 45 Pe RGO 60 AZR DGD Pb Run Tht Not Run Agd hei 61 and 75 80

61u 61u

Agd Run

Run

BU5 <211>

351 <212>

DNA <213>

ZERO

Bar! els <400>

Running dddhdsads hddhdsads

Hdddasadad dtdaaaaads ssddddadgs hshdaadahs

BO hsskhdhaadd dkhkhskhddaha sadshkhhass adshasgdda hsddskhdddh dsdssadahd

120 sssdddaaad dsskhddadhd dahdddddahs ahshahsskhd dhdashkhshda hassadahas

180 address

Hssaaddssa ddhsassahs

Hsadssdasa aggssahsad sassdsshas

240 shdsadhdd dsadsskhdaa dddsskhsddas assdssshdh atkhaskhdhdd dhsdddddds

300 hashddhash hsdahhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh

351 <210>

<211>

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Noto Sartels <400>

and Wa1 C1p hei Wa1 C1p Running s1u A1a 01 and Wa1 5 10

1-У5 Ху5 РГО 61 у 61 and

- 44 011876

Zeg Bie love 11th twenty 5eg Suz love 61 y Zeg 25 61 y Tug Zeg Pb Tht thirty Zeg tug Tgr 11th 61 y 35 tgr ya1 Agd 61 η meth 40 Rgo 61 y love 61 y Bie 45 61and TGr meth C1u 11th fifty 11 e tug RGO 61 y Azr 55 Zeg A5r tNg Agd Tug 60 zeg Rgo zeg Pb 61 η 65 61 y 61 η wah! Tht Not 70 Zeg A1a AZR Ace zeg 75 11 e zeg TNG A1a tug 80 yee 61 η Tgr zeg Zeg 85 yee 1-U5 A1a Zeg A5r 90 Tht A1a meth tug tug 95 SU5 61 y zeg 61 y Zeg one hundred Tug Tgr Tug Pb Azr 105 yee Tgr 61y Agd 61 y 110 Tht yee Ya1 tNg Ya1 Zeg Zeg 115

<210> eleven <211> 324 <212> DNA <213> Noto zar! Epz <400> eleven

daaattdtdt tdasdsadts tssaddsass sdtstttdt stssadddda ststsstdsa dddssadtsa dadtdttads adsadsTssg tadsstddta sstddssadd stsssaddst sstsataTat ddtdsatssa dsadddssas dasaddttsa dtddsadtdd dtstdddasa dasttsasts TsassaTsad sstdaadatt ttdsadtdta TTastdhsad sddtstddgd dsTsatsatt sstdddassa aadtddagat saaa <210> 12 <211> 108 <212> _np <213> note zartepz <400> 12 < 51 and X1e ya1 yee TNg s! N zeg rgo s! U tg Bei zeg yen 5eg ¹ 5 10

C1i Agd A1a tg yei 5eg su5 Agd A1a Zeg 61l zeg Wa1 Zeg ²⁰ 25 zaadadssass60 ssadsadaaa120 tdddsatsssa180 sadastddad240 sasttsdsds 300

324

Рgo 61 у

Zeg Zeg

- 45 011876

Zeg yee A1a 35 tgr tug C1p 61l (-U5 40 Rgo 61y 61 p A1a Rgo 45 Agd yee yee 11th Tug fifty 61 y A1a Zeg zeg Agd 55 A1a TNG C1 y Not Rgo 60 A5r Agd RNe Zeg b1u 65 Zeg 61 y zeg 61 y tNg 70 Azr RNe TNG Bie TNG 75 And e 5eg Agd yee 61and 80 RGO 61and Azr pH A1a 85 Ya1 Tug tug suz 61 η 90 Agd Zeg 61 y 61y zeg 95 zeg RNe tNg RNe C1u one hundred RGO 61y TNG B5 Ya1 105 Azr 11 e Bz

<210> 13 <211> 351 <212> DNA <213> Noto 5ar1epz <400> 13 daddgdsads hsskhdhaadd sssddddaaad addssddhsskh sddsadhdda hashddddhash gddddhddhddddhhhhhhh

Hddadsadad saasgggass datddddahs ddhsassahs ddssgsddas dddssdhdds dhdaaaaads adskhaskhdda ahskhahsskhd Hsadssdasa assdssahdg assskhddhsa ssddddadhs Gsddskhdddh dsdaskhskhda adhssahsad ahhaskhdhdd ssdHsgsHad hskhdaadahs dsdssadahd hassadahas sassdsskhas HHsddddads ΐ

120

180

240

300

351 <210> 14 <211> 117 <212> PRT <213> Note zarlepz <400> 14

61 and one wah! 61p Bie Ya1 5 61 p 5eg 61 y A1a 61 and 10 Ya1 love love RGO 61 y fifteen 61 and Zeg yee Bz 11th twenty zeg Suz Bz 61y Zeg 25 61 y Tug AZP RNe tNg thirty zeg Tug Tgr 11th 61 y 35 Tgr \ / a1 Agd 61 p fur 40 Rgo 61y love 61 y Bie 45 61 and Bie fur 61 y 11th fifty 11th tug Rgo 61 y Azr 55 Zeg Azr TNG Agd Tug 60 zeg Rgo Zeg RNe

- 46 011876

61 l 65 s! u 61 p wah! Tht Bie 61 η Tgr zeg Zeg 85 <31 y zeg 61 y Zeg one hundred Tug wah! ΊΤ1Γ wah! 115 zeg zeg

<210>

Pe Zeg A1a A5r Bw5 5eg Pe Zeg Tht A1a tug 70 75 80 Bie | _ua A1a zeg A5r 90 Tht A1a fur Tug Tug 95 Su 5 Tgr Tug Pb A £ p yee Tgr (31 y Agd C1 y Tht yee 105 110

<211>

324 <212>

DNA <213>

but that

Zartepz <400> daaahhdhdh hdasdsadhs gssaddsass sgddgskhhgdh sssadddda aadadssass sshhsshdsa dddssadhsa dadhdhhads adhadsshashh hadsshddh ssadssadaaa

120 ssddddssadss ssssaddsh ssgsagahag dddhsahssa dsadddssas gdddsagssa

180 dasaddhhsa dhdddsadhdd dsskhddddas dashkhsasgs

Hsassahsad Sadhashddad

240 sshdaadahg ghdsadhdha hhaskhdhsad sddhshdddsd dhsahsahh sashhhhdsdds

300 sshddddassa aadhddahah saaa

324 <210>

<211>

108 <212>

PRT <213>

Noto ripened5 <400>

61 and 11th Ya1 Bie thr C1p zeg RGO 61 y thr yee Zeg yee Zeg RGO 61 y one 5 10 fifteen 61 and Agd A1a Tht yee Zeg Suz Agd A1a 5eg 61 η Zeg Ya1 Zeg zeg zeg twenty 25 thirty tug yee A1a 35 tgr tug 61l 61l love 40 Rgo boo 61l A1a Rgo 45 Agd yee yee 11th tug fifty 61 y A1a zeg run Agd 55 D1a Tht 61 y 11th Rgo 60 Abr Agd Pb 5eg 61 y Zeg 61 y Zeg 61 y thr A5r Pb thr yee Tht Not zeg Agd yee 61 and 65 70 75 80

- 47 011876

Рgo 61 and A5r Pye A1a 85 Wah! Tug tug Suz 61p 90 Agd Zeg b1u C1u 5eg Zeg 95 Rie Thr Pb 61 at Rgo one hundred 61 y Tht B5 Ya1 105 A5P 11 e 1-U5 <210> 17 <211> 447 <212> PRT <213> Boto nourishment

<400> 17

61 and one wah! 61 η ye Wa1 61 η 5 Zeg 61 y A1a 61 and Wa1 10 love love RGO 61 y fifteen 61 and 5eg yee Bw5 11 e Zeg Suz Bz 61 y Zeg 61 y Tug AZL RNe Tht Zeg tug twenty 25 thirty tgr 11th b1u 35 Tgr Ya1 Agd 61l meth 40 Rgo 61 y love 61 y yee 45 61 and Bie fur C1u Not 11th Tug Rgo 61 y AZR zeg A5r Tht Agd Tug zeg Rgo zeg pb fifty 55 60 C1P boo 61 p Ya1 Tht Not zeg A 1a AZR love 5eg 11th zeg Tht A1a Tug 65 70 75 80 Bei 61 η Tgr Zeg Zeg 85 yee Bw5 A1a 5eg AER 90 YOU A1a fur Tug Tug 95 Suz 61 y zeg 61 y Zeg Tug Pb tug Pb AZR Bie Tgr 61 y Agd 61 y Tht yee one hundred 105 110 wah! Tht Ya1 Zeg Zeg A1a Zeg Tht Bz 61 y Rgo Zeg Ya1 Pb Rgo yee 115 120 125 A1a Rgo 130 Zeg Zeg love zeg thr 135 Zeg b! u 61u Tht A1 a 140 A1a yee 61 y Suz yee Ya1 Bw5 AZR Tug Pb Rgo 61 and RGO Ya1 YOU Ya1 Zeg tgr Azp Zeg 14 5 150 155 160 61 y A1a yee Tht Zeg 61 y ya1 H13 thr Pb RGO A1a Wah! yee 61 p Zeg 165 170 175 zeg 61 y Bie tug zeg Bie zeg Zeg Ya1 wah! thr Ya1 RGO Zeg zeg zeg 180 185 190 yee 61 y Tht 61 p Tht tug X1e suz AZP Ya1 AZP H15 love Rgo zeg AZP 195 200 205 Tht love Ya1 AZR Bz Bz Wah! 61 and RGO Bw5 Zeg Suz AZR Bz Tht H15 210 215 220 Tht suz RGO RGO Su5 RGO A1a Rgo 61and Bie yee 61 y 61y Rgo Zeg Ya1 225 230 235 240

- 48 011876

Rie bei Pb RGO RGO Buz RGO Buz Azr TG Bei mega Not Zeg Agd 255 Tht 245 250 Rgo 01and Wah! Tht Su5 Wah! wah! Wah! A5r Ya1 Zeg H15 C1 and Azr RGO 61 and 260 265 270 H / a Buz Pb A5P Tgr Tug Wah! AZR 61 y wah! 61 and ya1 NT 5 A5P but! but Buz 275 280 285 Tht BU5 RGO Agd <51 and C1 and b1p Tug AZP Zeg Tht Tug Agd wah! Ya1 Zeg 290 295 300 wah! yee Tht wah! ooh NT 5 61 p AZR Tgr Bei A5L about! at Buz 01 and tug Buz 305 310 315 320 CPS love Wah! zeg A5P BUZ A1a Bei rgo but! but Rgo 11 e 61and Buz Tht Not 325 330 335 Zeg BU 5 but! but Buz b! at b! P Rgo Agd 61 and Rgo 61 l Wah! Tug Tht Bei rgo 340 345 350 RGO zeg Agd A5r C1 and Bei Tht Buz AZP 61l Ya1 Zeg Bei Tht Suz Bei 355 360 365 Ya1 BUZ O1u Pb tug Rgo 5eg A5r Not A1a Wah! with! and Tgr C1 and zeg AZP 370 375 380 61 y FROM! η Rgo FROM! and AZP AZL Tug BUZ Tht Tht Rgo RGO Wah! Bei AZR Zeg 385 390 395 400 A5r boo Zeg Pb Pb 405 Bei tug Zeg Buz Bei 410 Tht Wah! A5r Buz Zeg 415 Agd Tgr b! p C1 p 01 y A5P wah! Pb zeg Su5 Zeg Wah! mega NT 5 01 and but! but Bei 420 425 430 ΗΊ 5 AZP ΗΊ5 Tug Tht 61 η Buz Zeg Bei Zeg Bei Zeg RGO C1u love 435 440 445 <210> eighteen <211> 215 <212> PRT <213> But that nourishment

<400>: 1.8 61 and Not wah! Bei Tht 61 η Zeg RGO C1u Tht Bei zeg Bei Zeg RGO 01 y one 5 10 fifteen 61 and Agd but! but Tht Bei zeg suz Agd but! but Zeg 01 η Zeg Wah! Zeg Zeg Zeg twenty 25 thirty Tug Bei A1a 35 tgr Tug 01 p 01 p love 40 Rgo 61y 01 p but! but Rgo 45 DGD yee Bei Not tug 01y but! but Zeg zeg Agd A1 a Tht 61 y •Not RGO Azr Agd Pb Zeg

55 60

- 49 011876

61 y 65 Run 61 y Run 61y Tht 70 Azr Pb Tht Bie Tht 75 And e Run Agd yee 61 and 80 Rgo 61 and AZR pb A1a 85 wah! Tug Tug Suz 61 p 90 Agd Run 61 y 61 y zeg 95 Run Pb Tht Pb 61 y one hundred RGO 61 y Tht B5 Ya1 105 Azr Not love Agd Tht 110 Ya1 A1 a A1a RGO Run 115 wah! ₽ Not Pb Rgo 120 Rgo Run A5r 61 and 61 η 125 Bie love Run 61 y Tht 130 A1a Run Ya1 wah! suz 135 yee yee Azp AZP Pb 140 tug Rgo Agd 61 and A1a 145 Bw Ya1 61 p Tgr love 150 Ya1 AZR AZP A1a yee 155 61η Run boo AZP Run 160 . 61 η b1i Run wah! Tht 165 61 and 61 η Azr Run. SU5 170 Azr Run Tht Tug Run 175 yee Zeg Run Tht bei 180 Tht yee Run love A1a 185 Azr Tug 61and love H13 190 B5 Ya1 Tug A1a Suz 195 61 and Ya1 Tht ΗΪ5 61 η 200 b1u yee Run Run Rgo 205 Ya1 Tht Bz

Running Rye Azp Agd C1u b1i Suz

215

<210> nineteen <211> 447 <212> PRT <213> But that <400> nineteen

61 and one Wah! 61 η Bie wah! 5 61 p Run 61 y A1a C1 and 10 Wah! love love Rgo 61 y fifteen 61 and Run Bie love 11th twenty Run suz bue 61 y Run 25 61y Tug Run Pb Tht thirty Run Tug tgr Not 61 y 35 Tgr wah! Agd b! p meth 40 RGO 61 y (-U5 61 y yee 45 61 and Tgr meth 61y Not fifty Not Tug RGO 61 y AZR 55 Run AZR Tht Agd Tug 60 Run Rgo Run Pb 61 p 65 b1u 61p ya1 Tht Not 70 Run A1a AZR B _y 5 Run 75 Not Run Tht A1a Tug 80 yee 61 p Tgr Run Run 85 Bie love A1a Run Azr 90 Tht but! but meth tug Tug 95 Suz 61 y Run 61y Run one hundred Tug tgr Tug Pb Azr 105 Bie tgr 61 y Agd 61y 110 Tht yee

- 50 011876

Ya1 you Ya1 115 5eg 5eg A1a Zeg Thr 120 bz C1u rgo zeg wah! 125 Pb Rgo yee A1a Rgo Zeg Zeg love Zeg Tht Zeg C! U b! U thr d1a A1a yee 61 y suz 130 135 140 yee wah! love Azr Tug Pb Rgo 61 and rgo Ya1 Tht Ya1 Zeg Tgr AZP Zeg 145 150 155 160 61 y but! but yee Tht Zeg s! u Ya1 NT 5 thr Pb RGO but! but Wah! yee 61 p zeg 165 170 175 Zeg 61y yee tug Zeg yee Zeg Zeg Wah! wah! YOU Wah! RGO 5eg Zeg Zeg 180 185 190 yee 61 y Tht 61l Tht tug 11th suz AZL Wah! A5L NTZ Bz RGO Zeg AZP 195 200 205 Tht Bz Wah! Azr love love Wah! 61 and RGO love Zeg suz AZR Bw5 Tht NT 5 210 215 220 Tht Suz Rgo Rgo Suz RGO A1a Rgo 61 and yee yee 6! at 61 y RGO zeg wah! 225 230 235 240 pke yee Pb RGO RGO love RGO love AZR Tht yee Meg 11th zeg Agd YOU 245 250 255 Rgo 61 and wah! Tht Suz Ya1 wah! Wah! AZR Ya1 Zeg ΗΊ 5 61 and Azr RGO 61 and 260 265 270 Ya1 love Pb 275 AZL Tgr Tug wah! AZR 280 61 y Wah! 61 and Ya1 NT 5 285 AZP A1a love thr love 290 RGO DGD 61 and 61 and 61 p 295 Tug AZP Zeg YOU Tug 300 DGD ya1 wah! Zeg wah! yee thr Ya1 Bie H15 61 p Azr Tgr yee A5P 61 y love 61 and tug love 305 310 315 320 Su 5 B5 Wah! Zeg AZP 325 love A1a yee RGO A1a 330 Rgo 11 e 61 and Bz Tht 335 11th Zeg love A1 a kU5 61 y 61l RGO Agd 61 and RGO 61l Ya1 tug YOU yee RGO 340 345 350 RGO Zeg Agd A5r 61 and Bie thr Bz AZP 61 p wah! Zeg Bie YOU Su5 yee 355 360 365 wah! love 61 y Pb Tug Rgo Zeg AZR 11th A1a Ya1 61 and Tgr 61 and zeg AZP 370 375 380 from! at 61 l Rgo 61 and AZL AZP Tug love Tht thr Rgo Rgo Ya1 Bie AZR Zeg 385 390 395 400 A5P 61 y zeg Pb Pb 405 Bie Tug Zeg love yee 410 Tht Wah! Azr love 5eg 415 DGD Tgr 61 p from! P 61y A5P Wah! Pb Zeg Suz Zeg wah! Me ΗΊ5 61 and A1a yee 420 425 430 NT 5 AZL NT5 Tug thr 61 η Bz Zeg Bie Zeg Bie Zeg Rgo boo Bz 435 440 445

- 51 011876

<210> twenty <211> 215 <212> PRT <213> But that <400> twenty

61 and one 11 e Ya1 yee Tht 5 61p Zeg Rgo 61 y TV g 10 Bie Zeg yee Zeg Rgo fifteen 61 y 61 and Agd A1a tig yee Zeg Suz Agd A1a Zeg 61 p zeg Wah! Zeg Zeg zeg twenty 25 thirty zeg Bie A1a 35 Tgr tug 61 p 61 η love 40 Rgo 61 y 61 p A1a Rgo 45 Agd yee Bie Not Tug fifty 61 y A1a zeg Zeg Agd 55 A1a Tht 61 y Not RGO 60 AZR Agd pb Zeg boo Zeg 61y Zeg 61 y Tht A5P rke Tht Bie TV g Pe Zeg Agd yee 61and 65 70 75 80 Rgo 61 about Azr Pb A1a Ya1 Tug Tug Suz 61 η Agd Zeg 61 y 61 y zeg zeg 85 90 95 Pb TV g Pb 61 y RGO 61 y Tht love Wah! AZR Not Bz Agd Tht ya1 A1a one hundred 105 110 A1a Rgo Zeg Wah! Pb Not Pb Rgo Rgo 5eg Azr 61 and 61 l Bie love zeg 115 120 125 61 y Tht 130 A1 a Zeg Ya1 Ya1 Suz 135 yee yee AZP AZP Pb 140 Tug RGO Agd 61 and A1a Bz Wah! 61 l Tgr Bw5 wah! Azr AZP A1a Bie 61P 5eg C1u AZP zeg 145 150 155 160 61 η 61 and Zeg Wah! Tht 61 and 61p A5P Zeg love Azr Zeg Tht Tug Zeg yee 165 170 175 Zeg Zeg thr Bie 180 Tht Bie Zeg love A1a 185 A5r Tug C1 and love H15 190 Bz Wah! Tug A1a suz 195 61 and wah! Tht H15 61 l 200 61y Bie zeg Zeg Rgo 205 wah! Tht love Zeg Rke A5P Agd 61 y 61 and SU5 210 215

<21O> 21 <211> 447 <212> PRT <213> noto sartels

- 52 011876 <4О0> 21

ST and one wah! b! p yee wah! 61l Zeg b! Y A1a e1 and Wa! love love RGO 61 y fifteen 61 and 5 10 Zeg yee Bz 11th twenty zeg Suz love 61 y Zeg 25 61 y tug AZP Riye TIG thirty zeg tug tgr Not 61 y 35 tgr Wah! Agd 61 p fur 40 Rgo 61 y Bw5 61 y yee 45 b1i tgr fur FROM! at Not Not Tug Rgo 61y AZR zeg Azr TIG DGD Tug 5eg Rgo zeg Riye fifty 55 60 61p C1u 61 η wah! TIG Not zeg A1a AZR love Zeg Not Zeg TIG but! but tug 65 70 75 80 yee C1p Tgr 5eg zeg 85 yee love A1a 5eg A5r 90 TIG A1a fur Tug tug 95 Suz b1u Zeg 61 y Zeg Tug Tgr tug Riye Azr Bie tgr 61 y Agd 61y TIG yee one hundred 105 110 wah! TIG Wah! Zeg Zeg A1a 5eg tyg B5 61 y Rgo zeg wah! Riye RGO yee 115 120 125 but! but Rgo 130 Zeg $ eh love zeg tyg 135 Zeg boo 61 y TIG A1a 140 but! but yee b1u Su 5 yee Wah! Bz A5r Tug Riye Rgo 61and rgo Ya1 TIG wah! zeg tgr AZP zeg 145 150 155 160 61 y BUT! but Bie TIG zeg from! at Ya1 H1 5 TIG Riye Rgo A1a Ya1 yee 61 p zeg 165 170 175 Zeg C1 y Bie Tug zeg Bie Zeg zeg Ya1 Ya1 TIG Wah! RGO Zeg 5eg zeg 180 185 190 Bie 61 y TIG 61 p TIG Tug Not Su5 AZL Ya1 Azl ΗΊ5 love Rgo Zeg AZL 195 200 205 TIG Bz Ya1 AZR bw5 love wah! 61 and RGO love Zeg Suz AZR bue tyg H15 210 215 220 TIG SU5 Rgo RGO suz Rgo A1a rgo 61and yee Bie C1u C1u RGO zeg ya1 225 230 235 240 Pb yee Pb RGO RGO love RGO love AZR TIG Bie Fur Not zeg DGD TIG 245 250 255 RGO 61 and Ya1 TIG 260 suz Wah! Wah! Ya1 AZR 265 Ya1 Run ΗΪ5 61 and Azr 270 RGO 61and Wah! love Pb AZP Tgr Tug wah! AZR b1u wah! 61 and Ya1 nt 3 AZP A1a love 275 280 285 TIG Bw5 Rgo Agd 61 and 61 and 61l Tug AZP zeg TIG Tug Agd wah! Ya1 zeg 290 295 300 ya1 yee TIG wah! yee ΗΊ5 61l Azr tgr yee Azp 61 y Bz 61 and Tug Bw5 305 310 315 320

- 53 011876

suz BUZ wah! Run A5P Buz A1a Bei Rgo A1a Rgo Not 61 and B5 Tht Not 325 330 335 Run BUZ d1a 1-Uz 61y 61 p RGO Agd 61 and Rgo 61 η Ya1 tug Tht Bei Rgo 340 345 350 RGO Run Agd AZR C1c Bei Tht Boo5 AZP 61 η Ya1 Run Bei Tht Suz Bei 355 360 365 wah! Buz C1u Pb tug RGO Run AZR Not A1a Ya1 C1 and Tgr 61 and Run AZP 370 375 380 61 y C1 η RGO C1 and AZP Azp Tug BUZ Tht Tht Rgo Rgo Ya1 Bei AZR Run 385 390 395 400 AZR 61 y 5eg Pb Riye 405 Bei Tug 5eg Buz Bei 410 Tht wah! AZR Buz Run 415 Agd tgr 61 p 61 η C1u AZP wah! Pb Run Suz Run Ya1 mega H15 61 and A1a Bei 420 425 430 ΗΊ5 AZP ΗΊ5 Tug Tht 61 p Buz Run Bei Run Bei Run RGO 61y Buz 435 440 445

<210> 22 <211> 215 <212> LRT <213> Noto Zartez <400> 22

61 and one Not Ya1 Bei thr 5 61 η Run b1i Agd A1a Tht twenty Bei Run suz Tug Bei A1a 35 Tgr Tug 61 η 61 η Not Tug fifty C1u A1a Run Run Agd 55 61 y 65 Run C1u Run 61 y Tht 70 Azr Rgo 61 and Azr Pb A1a 85 wah! Tug Pb thr Pb boo one hundred RGO 61 y Tht A1a Rgo Run 115 Ya1 Pb Not Pb C1u thr 130 A1a Run Wah! Ya1 &

Rgo 61 y YOU Bei Run Bei Run Rgo 61 y 10 fifteen Agd A1a Run 61 η Run Ya1 Run Run Run 25 thirty Buz 40 Rgo 61 y 61 η A1a Rgo 45 Agd Bei Bei A1a Tht 61 y Not RGO 60 Azr Agd Pb Run Pb thr Bei thr Not Run Agd Bei 61 and 75 80 Tug suz 61 η Agd Run 61 y C1u Run Run 90 95 Buz Wah! 105 Azr Not Buz Agd Tht 110 ya1 A1a rgo Rgo Run AZR 61 and 61 η Bei Buz Run 120 125 Bei Bei AZP A $ P Pb Tug RGO Agd C1 and 140

- 54 011876

ah a Bz wah bchp tgr love MAH A5r AZP A1a Bie C1p zeg <31u Azp zeg 145 150 155 160 bchp SCH Zeg wah Tht bchi <31 η A $ p Zeg Bz Azr Zeg Tht Tug Zeg Bie 165 170 175 Zeg Zeg Tht yee 180 Tht Bie Zeg love A1a 185 AZR tug C) and Bz H15 190 (-U5 Wha Tug dcha Suz <31 and Wha Tht ΗΪ5 C1p 61 y yee Zeg Zeg RGO Wha Tht Bz 195 200 205 Zeg Pb AZP Agd bcu SCH Suz 210 215

<210> 23 <211> 24 <212> DNA <213> Artificial sequence <22О>

<223> Oli gon cleotide primer for PCR <220>

<221> rt5c_ / eaTige <222> 08) .. (23) <223> η Ί5 a,, c, ΐ, og d <400> 23 dddssdatad dssgssapppp πηητ <210> 24 <211> 21 <212> DNA < 213> Artificial sequence <220>

<223> Oligonucleotypic polymerization PIR <400> 24 dddddhsadds hdddaaskhdad d <210> 25

- 55 011876 <211> 19 ^<212> DNA ^<21 3> Artificial sequence <22O>

<223> _{ABOUT L11HONUCLE (SUMMARY of the} 1st payera PIR <400> 25 tddaddst dassasasd <210> 26 <211?. 48 <212> DNA <213> Artificial sequence <22О>

<22 3> Oligo nucleotide primer for PCR <400> 26 asaaaaads ttsgadassa ssagddaaas sssadsssad stgststt <210> 27 <211> 33 <212? DNA <213> Artificial sequence <22O>

<223> Oligonucleotide primer for PCR <400> 27 stgdTsdast saasassssss sssgdttdaa dss <210> 28 <211> 44 <212> DNA <213> Artificial sequence

- 56 011876 <22O>

<223> Oligonucleotide polymella for SPR <400> 28 sadsadaads hgsgadassa ssa-sddddgs aassdesats stsd <210> 29 <211> 42 <212> DNA <213> Artificial sequence <220>

<22 3> Oligonucleotyl paymep for PIR <400> 29 sggddhdddad dsasgadada sddgdassad dddhdssasdd ss <210> 30 <211> 117 <212> PRT <213> Noto zurtz <400> 30

61 0 wah! 61p sei Ya1 61p Zeg 61y A1a 61 and Ya1 1uz 1-U5 Rgo boo 61and one 5 10 fifteen Zeg yee $ 1 11th twenty Zeg Suz Bz C1u Zeg 25 61 y Tug Zeg RNe YOU thirty Zeg Tug tgr Pe 61y 35 tgr Ya1 Agd 61l meth 40 Rgo 61y Bw5 61 y yee 45 with! and Tgr mega 61 y 11th fifty Not Tug Rgo 61 y AZR 55 Zeg A $ p TNG Agd tug 60 zeg Rgo Zeg Pb 61 η 65 <31 y 61 η Ya1 TNG 11 e 70 Zeg A1a Azr love Zeg 75 And e zeg TNG A1a tug 80 yee C1p Tgr Zeg Zeg 85 Yeah B5 A1a Zeg AZR 90 TNG A1a mega tug Tug 95 Suz 61 y zeg 61 y Zeg one hundred Tug Tgr Tug RNe Azr 105 1st Agd 61 y DGD 61 y 110 TNG yee Uat Tht wah! Zeg Zeg

115 <210> 31

<211> 109 <212> PRT <213> Noto Zarzepz

<400>: 31 61 and Not Ya1 Bie Tht 61 η Zeg Rgo 61 y Tht Bie Zeg Bie Zeg RGO b1u one 5 10 fifteen 61 and Agd A1a tNg Bie 5eg Suz Agd A1a zeg 61 η Zeg 11th Not Zeg Zeg twenty 25 thirty Tug yee A1a tgr tug 61 η 61 η 1-U5 Rgo 61 y 61 η TNG Rgo Agd Bie yee 35 40 45 Not tug fifty 61y ya1 Zeg Zeg Agd 55 A1a Tht 61y Not RGO 60 AZR Agd Pb eeg 61 y Zeg 61 y zeg 61 y Tht AZR Pb thr Bie Tht Not Tht Agd yee 61 and 65 70 75 80 RGO 61 and AZR Pb A1a Ya1 Tug tug suz 61 η 61p Tug 61 y Azp Zeg Pb 85 90 95 meth tug TNG Pb boo 61p 61 y Tht love Bie 61and Not love one hundred 105

<210> 32 <211> 447 <212> PRT

<213> zero charge ΊβΠ3 <400> 32 61 and Ya1 61p Bie νβΐ 61 η zeg 61 y A1a 61 and Ya1 Bz Bz RGO 61y 61 and one 5 10 fifteen Zeg yee Bz Not twenty Zeg Suz love 61 y zeg 25 61 y Tug zeg pH thr thirty Zeg Tug tgr Not 61 y 35 tgr Ya1 Agd 61 p meth 40 Rgo 61y Bz 61 y Bie 45 61 and tgr meth b1u Not fifty Not tug Rgo 61y Azr 55 Zeg AZR TNG Agd Tug 60 Zeg RGO zeg Pb 61 p 65 61 y 61 p wah! tNg Not 70 Zeg A1a A5r love Zeg 75 Not zeg Tht A1a tug 80 Bie 61 l Tgr Zeg zeg 85 yee love A1a Zeg Azr 90 thr A1a meth tug tug 95 Suz 61 y Zeg 61 y zeg Tug tgr Tug Pb AZR yee Agd 61 y Agd 61 y tNg Bie

- 58 011876

one hundred 105 110 wah! thr UAZ Run Run AZA Run Tht love b1u RGO Run ya1 pb Rgo yee 115 120 125 AZA RGO Run Run love Run Tht Run 61y 61y Tht AZA A1a yee b1u suz 130 135 140 yee UAZ Iuz A5r Tug Pb rgo 61 and Rgo UAZ TNG UAZ Run tgr AZP Run 145 150 155 160 61 y A1a Bie thr Run 61 y wah! NTZ Tht Pb Rgo AZA wah! yee 61 P Run 165 170 175 5eg C1u yee Tug Run yee Run Run Ya1 Ya1 Tht UAZ RGO zeg Run Run 180 185 190 yee 61 y Tht 61 p Tht Tug 11 e Su AZP Ya1 AZP H! 3 love RGO Run Azp 195 200 205 tNg 210 UAZ dzr love love Ya1 215 61 and RGO Bz Run Su 5 220 AZR love thr H15 Tht Suz Rgo RGO Suz Rgo AZA Rgo 61 and yee Bie 61y 61 y RGO Run Ya1 225 230 235 240 Pb yee pb RGO Rgo love RGO love Azr thr yee meth 11th Run Agd Tht 245 250 255 Rgo bZi UAZ Tht Suz UAZ UAZ UAZ AZR UAZ Run ΗΊ 5 61 and AZR RGO 61 and 260 265 270 UAZ CPS Pb A5P Tgr Tug UAZ a * P 61 y Ya1 61 and wah! ΗΊ5 AZP A1a Bz 275 280 285 Tht love Rgo lgd C1 and 61and 61 η tug AZP Zeg Tht Tug Agd UAZ Ya1 Run 290 295 300 UAZ yee Tht Wah! yee H15 bzp Azr tgr yee AZP 61y love bZi tug love 305 310 315 320 suz love UAZ Run Azp 325 love AZA yee RGO AZA 330 Rgo 13th 61 and love Tht 335 11th Zeg love AZA "-U5 61 y 61 p RGO Agd bZi Rgo 61 η UAZ tug Tht yee Rgo 340 345 350 RGO zeg lgd AZR bZi yee Tht love AZP bzp Ya1 Zeg yee TNG Suz Bie 355 360 365 UAZ suz bzu Pb Tug RGO Run Azr 11 e A1a Ya1 61 and tgr 61 and Run Azp 370 375 380 C1u 385 bzp Rgo 61 and A5P A5P 390 Tug B5 Tht Tht RGO 395 RGO Ya1 Bie AZR Run 400 A5r bzu Run pb Pb yee tug Run love yee Tht Ya1 Azr Bz Run Agd 405 410 415 Tgr bzp bzp bzu AZP Ya1 pb Run suz zeg UAZ meth ΗΪ3 b! and A1a Bie 420 425 430 H! 5 Azp H13 Tug Tht bzl love Run Bie Zeg yee Run RGO 61 y love

- 59 011876

435 440 445 <210> 33 <211> 216 <212 " PRT <213> Noto Sartels

<400> 33

b! and one Not Wah! yee Tht 5 b! p Run RGO 61 and Agd A1a Tht twenty yee Run suz Agd Tug yee A1 a 35 Tgr tug 61 η 61 η love 40 Not tug fifty 61 y Wah! Run Run Agd 55 A1a 61 y 65 Run 61 y Run 61 y thr 70 AZR Pb RGO FROM! and A5P Pb but! but 85 wah! tug Tug meth Tug Tht Pb one hundred 61 y C1p 61 y Tht A1a A1 a Rgo 115 Run Ya1 Pb 11 e Pb 120 Run 61y 130 Tht but! but Run Wah! Wah! 135 Suz <31 and 145 A1a love Wah! 61 η tgr 150 Bw5 wah! Run 61 η 61 and Run Wah! 165 Tht <31 and 61 η yee Run Run thr 180 yee Tht yee Run wah! Tug but! but 195 Suz 61 and Wah! Tht ΗΊ5 200 Bw5 Run 210 Pb Azp Agd C1u 61and 215 SU5 <210> 34 <211> 5 <212> PRT <213> But that sartels

61 y Tht Bie Run Bie Run Rgo 61 y 10 fifteen but! but Run 61 η Run Not Not Run Run 25 thirty Rgo 61y 61 p Tht Rga Agd yee yee 45 Tht 61 y Not RGO 60 AZR Agd Pb Run YOU yee Tht Not Tht Agd yee 61 and 75 80 Suz 61 p 61 p Tug 61U AZP Run Pb 90 95 love yee b! and Not love Agd Tht wah! 105 but

Rgo Rgo Run dzr C1i 61p Lye L5

125 Bie yee AZP AZL 140 Pb Tug RGO Agd Azr Azp A1a yee 61p Run 61 y AZP 155 160 Azr Run Bw5 A5P Run Tht Tug Run 170 175 love A1a Azr tug 61 and love H15 love 185 190 61 η 61y Bie Run Run Rgo yeah) Tht 205

- 60 011876

<400> 34 zeg Tug tgr 11e b! y one 5 <210> 35 <211> 17 <212> PRT <213> Noto Zartezz

<400> 35

Not 11th Tug rgo 61y Azr Zeg A5r tg Agd Tug zeg Rgo Zeg Rb 61l one 61y 5 10 fifteen <210> 36 <211> 8 <212> PRT <213> noto salage <400> 36 01 at Zeg  Tug Rye Tug Rye Azr yee one 5 <210> 37 <211> 8 <212> PRT <213> Noto Zar1ep5

<400> 37 61u zeg one • Tug Tgr Tug Rb Azr Uye <210> 38 <211> 12 <212> PRT <213> Noto zar1el5

- 61 011876 <400> 38

Agd A1a Running 61 η Running ya! Running Running Running Tug yei d1a 1 5 u <210> 39 <211> 12 <212> PRT <213> kot zar! En5 <400> 39

Agd A1a Run C1n Run ya1 Run Run Run Run Lea A1a 1 510 <210> 40 <211> 12 <212> PRT <213> Note salary <400> 40 dgd A1a Run 61 η Run 11е 11 е Run Run Tugye A1a 1 5yu <210> 41 <211> 7 <212> PRT <213> not a charge? EPZ <400> 41 at A1a Run Run dgd d! and Tg <210> 42 <211> 7 <212> PRT <213> Note salary

- 62 011876

<400> 42 <51 at Wa! one Run Run Agd a! A Tg <210> 43 <211> nine <212> PRT <213> but that nourishment

<400> 43 b! p Agd Running C1u C1u Running Running Pb one 5 <210> 44 <211> 10 <212> PRT <213> Noto Zartezz

<400> 44 <51n 61 η thug C1y azp Run PbE MeT Thug tb 1,510 <210> 45 <211> 24 <212> DNA

<213> Noto Salage <400> 45 ddddadstasg thtasttsda Sehe 24 <210> 46 <211> 24 <212> DNA <213> noto zar £ epz <400> 46 ddddadstast ddhasttsda Tess 24

- 63 011876

<21O> 47 <211> 27 <212> DNA <213> noto zartezz <400> 47

sadsddhhhhh dhddhhsats ahhhsash <210> 48 <211> 9 <212> PRT <213> Noto zarepz <22O>

<221> and EE <222> (2) .. (9) <223> x is any amino acid <400> 48

Suz haa haa haa haa haa haa haa <210> 49 <211> 5 <212> PRT <213> Noto zarlezz

<400> 49 yei C1 one and Tgr 11e 61u <210> fifty <211> 4 <212> PRT <213> Noto Salage

- 64 011876

<220> <221> <222> <223> lowland ¢ 3),. (3) x is any amino acid <400> fifty

Tgr 01 y haa 01u <210> 51 <211> 4 <212> PRT <213> Noto zartepz <220>

<221> si5yeE <222> (3) .. (3) <223> ^x is any amino acid <400> 51 pye c1u haa c1u <210> 52 <211> 20 <212> _prt <213> Noto zaraptes <400> 52 meg <; 1and YOU dzr tg Bei Bey Betz tgr wa! Bey Bey _willows and Tgr wa! rgo * *> 1015

01y Zeg YOU 61y <210> 53 <211> 26

- 65 011876

<212> PRT <213> zero nourishment <400> 53 Μβΐ A1 a Tb c! zeg agd tg zeg b Bie Ley A1a Rwe 61 at Eye Yee one 5 10 fifteen

Suz yee ₽go tgr yee s! η c1 and c! 5g A1a

2025 <210> 54 <211> 19 <212> PRT <213> Noto zartz <400> 54 megabytes! Zeg tkg l1a Ne yee A1a yee yee yee A1a Wa! ёи αΐη б! у 15 1015 уа! Suz a! A <210> 55 <211> 20 <212> PRT

<213> Zero Zartez <400> 55 Mes 61 and tkg Rgo D1a 61p yeeee yee rke yee yee Еи и Т г г ье р р го го one 5 10 fifteen Azr Thg tkg 61 y twenty <210> 56 <211> 351 <212> DNA <213> But that zar еп epz

<400> 56 ddddgdsads sdddsdsadss sdddsadad ddaaaaads ssddddddss sssdaadass 60

- 66 011876 hsshddhaadd dhhhssddaha sadshhhass adshaskhdda hddskhdddh dsddssadahd

120 sssdddaaad dsskhddadhd dahdddddahs ahshassshd dHdasHssda hassadahas

180 addressssdhssh hssaaddssa ddhsassahs hsadssdasa addssahsad sassdsshas

240 shdsadhdd dsadsskhdaa ddsskhsddas assdssahdh ahhaskhdhdd dhhsdddddads

300 hashddhahh hsdahhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh

351 <210>

<211?

<212>

327

DNA <213>

fingernail nartepz <400> daaahhdhdh hdasdsadhs sssaddsass sddhhhhhh shssadddda aadadssass sxhsshdsa dddssadhsa dadhahhahs adshadsshash hadsshddh ssadsadaaa

120 ssshddssada sssssaddskh skhsakhskhah dddtdhahssa dsadddssas hddsahsssa

180 dasaddhhsa dhddsadhdd dhskhdddasa dashkhsasts hsassahsas sadasgddad

240 sstdaadahh hhdsadhdha hhaskhdhsad sadhahddha askhsahhsah dhasaskhhhh

300 dddssadddda ssaadskhdda dahsaaa

327

Claims (55)

CLAIM 1. An isolated antibody containing the complementarity determining region 3 (ί.ΌΕ3) of the heavy chain having an amino acid sequence comprising at least 7 amino acids 8EC ΙΌ N0: 36 in the same order and spatial arrangement as they are located in 8EC ΙΌ N0: 36 capable of specifically binding to IΡN-γ. 2. The antibody according to claim 1, including ί.ΌΒ3 light chain having an amino acid sequence representing: (a) an amino acid sequence comprising at least 8 amino acids 8EC ΙΌ N0: 43 in the same order and spatial arrangement as they are located in 8EC ΙΌ N0: 43; or (b) an amino acid sequence comprising at least 9 amino acids 8EC ΙΌ N0: 44 in the same order and spatial arrangement as they are located in 8EC ΙΌ N0: 44. 3. The antibody according to claim 2, comprising one or more of the JUICE! a heavy chain having the amino acid sequence of 8EC ΙΌ N0: 34; SEC2 heavy chain having the amino acid sequence of 8EC ΙΌ N0: 35; SEC3 heavy chain having the amino acid sequence of 8EC ΙΌ N0: 36 or 8EC ΙΌ N0: 37; Light chain CEC1 having the amino acid sequence of 8EC ΙΌ N0: 38, 8EC ΙΌ N0: 39 or 8EC ΙΌ N0: 40, and / or light chain CEC2 having the amino acid sequence of 8EC ΙΌ N0: 41 or 8EC ΙΌ N0: 42. 4. The antibody according to claim 2, where ί.ΌΚ3 of the heavy chain has 8EC ΙΌ N0: 36, and CEC3 of the light chain has 8EC ΙΌ N0: 43. 5. The antibody according to any one of claims 1 to 4, containing the amino acid sequence of the variable region of the heavy chain, at least 80% identical to 8EC ΙΌ N0: 6, 8EC ΙΌ N0: 10, 8EC ΙΌ N0: 14 and / or 8EC ΙΌ N0 : 30, where the comparison of the amino acid sequence of the heavy chain with 8EC ΙΌ N0: 6, 8EC ΙΌ N0: 10, 8EC ΙΌ N0: 14 or 8EC ΙΌ N0: 30 covers at least 50 amino acids. 6. The antibody according to claim 5, where the amino acid sequence of the variable region of the heavy chain is at least 90% identical to 8EC ΙΌ N0: 6, 8EC ΙΌ N0: 10, 8EC ΙΌ N0: 14 and / or 8EC ΙΌ N0: 30. 7. The antibody according to claim 6, where the amino acid sequence of the variable region of the heavy chain is identical to 8ES) ΙΌ N0: 6, 8EO ΙΌ N0: 10, 8EO ΙΌ N0: 14 or 8EO ΙΌ N0: 30. 8. An antibody according to any one of claims 1 to 7, containing the amino acid sequence of the variable region of the light chain at least 80% identical to 8EC Ц N0: 8, 8EC ΙΌ N0: 12, 8EC ΙΌ N0: 16 and / or 8EC ΙΌ N0 : 31, where the comparison of the amino acid sequence of the light chain with 8EC ΙΌ N0: 8, 8EC ΙΌ N0: 12, 8EC ΙΌ N0: 16 and / or 8EC ΙΌ N0: 31 covers at least 50 amino acids. - 67 011876 9. The antibody of claim 8, where the amino acid sequence of the variable region of the light chain is at least 90% identical to δΕρ ΙΌ ΝΟ: 8, δΕρ ΙΌ ΝΟ: 12, δΕρ ΙΌ ΝΟ: 16 and / or δΕρ ΙΌ ΝΟ: 31. 10. The antibody according to claim 9, where the amino acid sequence of the variable region of the light chain is δΕρ ΙΌ ΝΟ: 8, δΕρ ΙΌ ΝΟ: 12, δΕρ ΙΌ ΝΟ: 16 or δΕρ ΙΌ ΝΟ: 31. 11. The antibody according to any one of claims 1 to 4, containing the amino acid sequence of the heavy chain at least 80% identical to δΕρ ΙΌ ΝΟ: 17, δΕρ ΙΌ ΝΟ: 19, δΕρ ΙΌ ΝΟ: 21 and / or δΕρ ΙΌ ΝΟ: 32 where the comparison of the amino acid sequence of the heavy chain with δΕρ ΙΌ ΝΟ: 17, δΕρ ΙΌ ΝΟ: 19, δΕρ ΙΌ ΝΟ: 21 and / or δΕρ ΙΌ ΝΟ: 32 covers at least 50 amino acids. 12. The antibody according to claim 11, where the amino acid sequence of the heavy chain is at least 90% identical to δΕρ ΙΌ ΝΟ: 17, δΕρ ΙΌ ΝΟ: 19, δΕρ ΙΌ ΝΟ: 21 and / or δΕρ ΙΌ ΝΟ: 32. 13. The antibody of claim 12, wherein the amino acid sequence of the heavy chain is δΕρ ΙΌ ΝΟ: 17, δΕρ ΙΌ ΝΟ: 19, δΕρ ΙΌ ΝΟ: 21, or δΕρ ΙΌ ΝΟ: 32. 14. The antibody according to any one of claims 1 to 4 and 11-13, containing the amino acid sequence of the light chain at least 80% identical to δΕρ ΙΌ ΝΟ: 18, δΕρ ΙΌ ΝΟ: 20, δΕρ ΙΌ ΝΟ: 22 and / or δΕρ ΙΌ ΝΟ: 33, where the comparison of the amino acid sequence of the light chain with δΕρ ΙΌ ΝΟ: 18, δΕρ ΙΌ ΝΟ: 20, δΕρ ΙΌ ΝΟ: 22 and / or δΕρ ΙΌ ΝΟ: 33 covers at least 50 amino acids. 15. The antibody of claim 14, wherein the amino acid sequence of the light chain is at least 90% identical to δΕρ ΙΌ ΝΟ: 18, δΕρ ΙΌ ΝΟ: 20, δΕρ ΙΌ ΝΟ: 22 and / or δΕρ ΙΌ ΝΟ: 33. 16. The antibody according to clause 15, where the amino acid sequence of the light chain is δΕρ ΙΌ ΝΟ: 18, δΕρ ΙΌ ΝΟ: 20, δΕρ ΙΌ ΝΟ: 22 or δΕρ ΙΌ ΝΟ: 33. 17. The antibody according to claim 1, containing the amino acid sequence of the variable region of the heavy chain, which is: (a) an amino acid sequence at least 96% identical to δΕρ ΙΌ ΝΟ: 6; (b) an amino acid sequence at least 93% identical to δΕρ ΙΌ ΝΟ: 10; (c) an amino acid sequence at least 91% identical to δΕρ ΙΌ ΝΟ: 14; or (ά) an amino acid sequence at least 98% identical to δΕρ ΙΌ ΝΟ: 30; where the comparison of the amino acid sequence of the variable region of the heavy chain with δΕρ ΙΌ ΝΟ: 6, δΕρ ΙΌ ΝΟ: 10, δΕρ ΙΌ ΝΟ: 14 or δΕρ ΙΌ ΝΟ: 30 covers at least 50 amino acids. 18. The antibody according to 17, where the amino acid sequence of the variable region of the heavy chain is at least 95% identical to δΕρ ΙΌ ΝΟ: 10 or 95% identical to δΕρ ΙΌ ΝΟ: 14. 19. The antibody of claim 18, wherein the amino acid sequence of the variable region of the heavy chain is δΕρ ΙΌ ΝΟ: 6, δΕρ ΙΌ ΝΟ: 10, δΕρ ΙΌ ΝΟ: 14, or δΕρ ΙΌ ΝΟ: 30. 20. The antibody according to any one of paragraphs.17-19, containing the amino acid sequence of the variable region of the light chain, at least 80% identical to δΕρ ΙΌ ΝΟ: 8, δΕρ ΙΌ ΝΟ: 12, δΕρ ΙΌ ΝΟ: 16 and / or δΕρ ΙΌ ΝΟ : 31, where the alignment between the amino acid sequence of the light chain and δΕρ ΙΌ ΝΟ: 8, δΕρ ΙΌ ΝΟ: 12, δΕρ ΙΌ ΝΟ: 16, or δΕρ ΙΌ ΝΟ: 31 covers at least 50 amino acids. 21. The antibody according to claim 20, where the amino acid sequence of the variable region of the light chain is at least 90% identical to δΕρ ΙΌ ΝΟ: 8, δΕρ ΙΌ ΝΟ: 12, δΕρ ΙΌ ΝΟ: 16 and / or δΕρ ΙΌ ΝΟ: 31. 22. The antibody according to any one of paragraphs.17-21, where the amino acid sequence of the COB3 heavy chain includes at least 5 amino acids in δΕρ ΙΌ ΝΟ: 36 and / or δΕρ ΙΌ ΝΟ: 37 in the same order and spatial arrangement as they are located in δΕρ ΙΌ ΝΟ: 36 and / or δΕρ ΙΌ ΝΟ: 37. 23. The antibody according to claim 20 or 21, where the amino acid sequence of COB3 light chain includes at least 6 amino acids in δΕρ ΙΌ ΝΟ: 43 or δΕρ ΙΌ ΝΟ: 44 in the same order and spatial arrangement as they are located in δΕρ ΙΌ ΝΟ: 43 or δΕρ ΙΌ ΝΟ: 44. 24. The antibody according to claim 1, capable of inhibiting the biological activity of ΙΕΝ-γ human and ΙΕΝ-γ chimpanzee and not inhibiting the biological activity of ΙΕΝ-γ monkey cynomolgus and ΙΕΝ-γ mouse, and not able to inhibit the biological activity of the mutant variant ΙΕΝ-γ human where the histidine at position 19 is replaced by aspartate and / or where the serine at position 20 is replaced by proline. 25. The antibody according to paragraph 24, capable of inhibiting the biological activity of the mutant variant of the ΙΕΝ-γ monkey cynomolgus, in which histidine at position 19 replaces aspartate, serine at position 20 replaces proline and serine at position 65 replaces arginine. 26. The antibody according to claim 1, containing COB1 light chain, COB2 light chain, COB3 light chain, COB1 heavy chain, COB2 heavy chain and COB3 heavy chain, where COB1 of the heavy chain has the amino acid sequence δΕρ ΙΌ ΝΟ: 34; COB2 of the heavy chain has the amino acid sequence δΕρ ΙΌ ΝΟ: 35; COB3 of the heavy chain has the amino acid sequence δΕρ ΙΌ ΝΟ: 36 or δΕρ ΙΌ ΝΟ: 37; COB1 of the light chain has the amino acid sequence δΕρ ΙΌ ΝΟ: 38, δΕρ ΙΌ ΝΟ: 39 or δΕρ ΙΌ ΝΟ: 40; COB2 light chain has the amino acid sequence δ последовательностьρ ΙΌ ΝΟ: 41 or δΕρ ΙΌ ΝΟ: 42; COB3 light chain has the amino acid sequence δΕρ ΙΌ ΝΟ: 43 or δΕρ ΙΌ ΝΟ: 44. - 68 011876 27. The antibody of claim 26, wherein SEV3 of the heavy chain has an amino acid sequence of 8Ε0 ΙΌ ΝΟ: 36, SEV1 light chain has an amino acid sequence of 8Ε0 ΙΌ ΝΟ: 38, CMEA light chain has an amino acid sequence of 8Ε0 ΙΌ ΝΟ: 41 and SEV3 light chain has an amino acid sequence of 8Ε0 ΙΌ ΝΟ: 43. 28. The antibody according to p. 26 or 27, containing the amino acid sequence of the heavy chain at least 80% identical to 8ΕΟ ΙΌ ΝΟ: 17, 8ΕΟ ΙΌ ΝΟ: 19, 8ΕΟ ΙΌ ΝΟ: 21 and / or 8ΕΟ ΙΌ ΝΟ: 32, where a comparison of the amino acid sequence of the heavy chain with 8ΕΟ ΙΌ ΝΟ: 17, 8ΕΟ ΙΌ ΝΟ: 19, 8ΕΟ ΙΌ ΝΟ: 21 and / or 8ΕΟ ΙΌ ΝΟ: 32 covers at least 50 amino acids, and the amino acid sequence of the light chain is at least 80% identical to 8ΕΟ ΙΌ ΝΟ: 18, 8ΕΟ ΙΌ ΝΟ: 20, 8ΕΟ ΙΌ ΝΟ: 22 and / or 8ΕΟ ΙΌ ΝΟ: 33, where the amino acid sequence of the light chain is compared with 8ΕΟ ΙΌ ΝΟ: 18, 8ΕΟ Ό ΝΟ: 20, 8ΕΟ ΙΌ ΝΟ: 22 and / or 8ΕΟ ΙΌ ΝΟ: 33 spans at least 50 amino acids. 29. The antibody of claim 28, wherein the heavy chain amino acid sequence is at least 90% identical to 8ΕΟ ΙΌ ΝΟ: 17, 8ΕΟ ΙΌ ΝΟ: 19, 8ΕΟ ΙΌ ΝΟ: 21 and / or 8ΕΟ ΙΌ ΝΟ: 32 and the light amino acid sequence the chain is at least 90% identical to 8ΕΟ W ΝΟ: 18, 8ΕΟ ΙΌ ΝΟ: 20, 8ΕΟ ΙΌ ΝΟ: 22 and / or 8ΕΟ ΙΌ ΝΟ: 33. 30. The antibody according to clause 29, in which the amino acid sequence of the heavy chain has 8ΕΟ ΙΌ ΝΟ: 17, 8ΕΟ ΙΌ ΝΟ: 19, 8ΕΟ ΙΌ ΝΟ: 21 and / or 8ΕΟ ΙΌ ΝΟ: 32 and the amino acid sequence of the light chain has 8ΕΟ ΙΌ ΝΟ: 18, 8ΕΟ ΙΌ ΝΟ: 20, 8ΕΟ ΙΌ ΝΟ: 22 and / or 8ΕΟ ΙΌ ΝΟ: 33. 31. The antibody according to p. 26 or 27, containing the amino acid sequence of the variable region of the heavy chain, at least 80% identical to 8ΕΟ ΙΌ ΝΟ: 6, 8ΕΟ ΙΌ ΝΟ: 10, 8ΕΟ ΙΌ ΝΟ: 14 and / or 8ΕΟ ΙΌ ΝΟ: 30 where the comparison between the amino acid sequence of the variable region of the heavy chain and 8ΕΟ ΙΌ ΝΟ: 6, 8ΕΟ ΙΌ ΝΟ: 10, 8ΕΟ ΙΌ ΝΟ: 14 and / or 8ΕΟ ΙΌ ΝΟ: 30 covers at least 50 amino acids; and the amino acid sequence of the variable region of the light chain, at least 80% identical to 8ΕΟ ΙΌ ΝΟ: 8, 8ΕΟ ΙΌ ΝΟ: 12, 8ΕΟ ΙΌ ΝΟ: 16 and / or 8ΕΟ ΙΌ ΝΟ: 31, where the comparison between the amino acid sequence of the variable region of the light chain and 8ΕΟ ΙΌ ΝΟ: 8, 8ΕΟ ΙΌ ΝΟ: 12, 8ΕΟ ΙΌ ΝΟ: 16 and / or 8ΕΟ ΙΌ ΝΟ: 31 covers at least 50 amino acids. 32. The antibody of claim 31, wherein the amino acid sequence of the variable region of the heavy chain is at least 90% identical to 8ΕΕ) ΙΌ ΝΟ: 6, 8ΕΟ ΙΌ ΝΟ: 10, 8ΕΟ ΙΌ ΝΟ: 14 and / or 8ΕΟ ΙΌ ΝΟ: 30 and the amino acid sequence of the variable region of the light chain is at least 90% identical to 8ΕΟ ΙΌ ΝΟ: 8, 8ΕΟ ΙΌ ΝΟ: 12, 8ΕΟ ΙΌ ΝΟ: 16 and / or 8ΕΟ ΙΌ ΝΟ: 31. 33. The antibody of claim 32, wherein the amino acid sequence of the variable region of the heavy chain is 8ΕΟ ΙΌ ΝΟ: 6, 8ΕΟ ΙΌ ΝΟ: 10, 8ΕΟ ΙΌ ΝΟ: 14 or 8ΕΟ ΙΌ ΝΟ: 30 and the amino acid sequence of the variable region of the light chain is 8ΕΟ ΙΌ ΝΟ: 8, 8ΕΟ ΙΌ ΝΟ: 12, 8ΕΟ ΙΌ ΝΟ: 16 or 8ΕΟ ΙΌ ΝΟ: 31. 34. The antibody according to claim 1, containing: (a) the amino acid sequence of the variable region of the heavy chain, including 8ΕΟ ΙΌ ΝΟ: 6, 8ΕΟ ΙΌ ΝΟ: 10, 8ΕΟ ΙΌ ΝΟ: 14 or 8ΕΟ ΙΌ ΝΟ: 30, or a fragment of any of these sequences, and (b) the amino acid sequence of the variable region of the light a chain including 8ΕΟ ΙΌ ΝΟ: 8, 8ΕΟ ΙΌ ΝΟ: 12, 8ΕΟ ΙΌ ΝΟ: 16 or 8ΕΟ ΙΌ ΝΟ: 31, or a fragment of any of these sequences. 35. The antibody according to clause 34, containing: (a) the amino acid sequence of the variable region of the heavy chain 8ΕΟ ΙΌ ΝΟ: 6, 8ΕΟ ΙΌ ΝΟ: 10, 8ΕΟ ΙΌ ΝΟ: 14 or 8ΕΟ ΙΌ ΝΟ: 30; and (b) the amino acid sequence of the variable region of the heavy chain 8ΕΟ ΙΌ ΝΟ: 8, 8ΕΟ ΙΌ ΝΟ: 12, 8ΕΟ ΙΌ ΝΟ: 16 or 8ΕΟ ΙΌ ΝΟ: 31. 36. The antibody according to clause 35, containing 8ΕΟ ΙΌ ΝΟ: 6 and 8ΕΟ ΙΌ ΝΟ: 8. 37. The selected antibody containing the amino acid sequence of the variable region of the light chain, which is: (a) an amino acid sequence of at least 97% identical to 8ΕΟ ΙΌ ΝΟ: 8; (b) an amino acid sequence at least 96% identical to 8ΕΟ ΕΟ ΝΟ: 12; (c) an amino acid sequence of at least 97% identical to 8ΕΟ ΙΌ ΝΟ: 16; or (b) an amino acid sequence at least 95% identical to 8ΕΟ ΙΌ ΝΟ: 31; - 69 011876 where the comparison of 8ЕО ΙΌ N0: 8, 8Е0 ΙΌ N0: 12, 8Е0 ΙΌ N0: 16, or 8Е0 ΙΌ N0: 31 with the amino acid sequence of the light chain comprises at least 50 amino acids capable of specifically binding to IEN-γ. 38. The antibody according to clause 37, where the amino acid sequence of the variable region of the light chain is at least 97% identical to 8Е0 ΙΌ N0: 8, 8Е0 ΙΌ N0: 12, 8Е0 ΙΌ N0: 16 or 8Е0 ΙΌ N0: 31. 39. The antibody according to § 38, where the amino acid sequence of the variable region of the light chain is 8E0 ΙΌ N0: 8, 8E0 ΙΌ N0: 12, 8E0 ΙΌ N0: 16 or 8E0 ΙΌ N0: 31. 40. The antibody according to clause 37, where the amino acid sequence of SBC3 light chain includes at least 6 amino acids in 8Е0 Ш N0: 43 or 8Е0 ΙΌ N0: 44 in the same order and spatial arrangement as they are located in 8Е0 ΙΌ N0: 43 or 8E0 ΙΌ N0: 44. 41. An antibody according to any one of claims 37-40, comprising the amino acid sequence of the variable region of the heavy chain at least 80% identical to 8E0 ΙΌ N0: 6, 8E0 ΙΌ N0: 10, 8E0 ΙΌ N0: 14 and / or 8E0 ΙΌ N0 : 30, where a comparison of the amino acid sequence of the variable region of the heavy chain with 8Е0 ΙΌ N0: 6, 8Е0 ΙΌ N0: 10, 8Е0 ΙΌ N0: 14 and / or 8Е0 ΙΌ N0: 30 covers at least 50 amino acids. 42. The antibody according to paragraph 41, where the amino acid sequence of the variable region of the heavy chain is at least 90% identical to 8Е0 ΙΌ N0: 6, 8Е0 ΙΌ N0: 10, 8Е0 ΙΌ N0: 14 and / or 8Е0 ΙΌ N0: 30. 43. The antibody according to paragraph 41 or 42, where the amino acid sequence of SBC3 of the heavy chain includes at least 5 amino acids in 8E0 ΙΌ N0: 36 and / or 8Е0 Ш N0: 37 in the same order and spatial arrangement as they are located in 8E0 ΙΌ N0: 36 and / or 8Е0 ΙΌ N0: 37. 44. The antibody according to any one of claims 1 to 43, which is a single chain antibody. 45. The antibody according to any one of claims 1 to 43, which is a fully human antibody. 46. The antibody according to any one of claims 1 to 43, which is a humanized antibody. 47. The selected polynucleotide encoding the antibody according to any one of claims 1 to 46. 48. A vector containing the polynucleotide according to item 47. 49. A host cell containing the vector of claim 48. 50. A method for producing an antibody capable of specifically binding to IEN-γ, comprising culturing a host cell according to claim 49. 51. A method of treating an IEN-γ mediated disease, comprising administering a therapeutically effective amount of an antibody according to any one of claims 1, 3-46. 52. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an antibody according to any one of claims 1 to 46. 53. A method of treating a disease mediated by IN-γ, comprising administering a composition according to claim 52. 54. The method of claim 51 or 53, wherein the ΙΕΚγ-mediated disease is AIDS, rheumatoid arthritis, including juvenile rheumatoid arthritis; inflammatory bowel disease, including ulcerative colitis and Crohn's disease; multiple sclerosis, Addison’s disease, diabetes (Ι type), epididymitis, glomerulonephritis, Graves disease, Guillain-Barré syndrome, chronic lymphomatous thyroiditis, hemolytic anemia, systemic lupus erythematosus (SLE), lupus nephritis, asthenic bulbar palsy psoriasis, p arthritis, atherosclerosis, erythropoietin resistance, transplant versus host, transplant rejection, autoimmune hepatitis-induced liver damage, biliary cirrhosis, alcohol-induced liver damage, Including alcoholic cirrhosis, rheumatism, sarcoidosis, scleroderma, Sjogren's syndrome, spondylarthropathy, including ankylosing spondylitis, thyroiditis or vasculitis. 55. The method according to item 54, where the disease mediated by IEn-γ is selected from the group consisting of rheumatoid arthritis, including juvenile rheumatoid arthritis, inflammatory bowel diseases, including ulcerative colitis and Crohn's disease, multiple sclerosis, systemic lupus erythematosus (SLE) lupus nephritis, psoriasis and psoriatic arthritis.